Immunogenicity of Constrained Monoclonal Antibody A32-Human Immunodeficiency Virus (HIV) Env gp120 Complexes Compared to That of Recombinant HIV ...
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JOURNAL OF VIROLOGY, May 2004, p. 5270–5278 Vol. 78, No. 10
0022-538X/04/$08.00⫹0 DOI: 10.1128/JVI.78.10.5270–5278.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Immunogenicity of Constrained Monoclonal Antibody A32-Human
Immunodeficiency Virus (HIV) Env gp120 Complexes Compared to
That of Recombinant HIV Type 1 gp120 Envelope Glycoproteins
Hua-Xin Liao,1* S. Munir Alam,1 John R. Mascola,2 James Robinson,3 Benjiang Ma,1
David C. Montefiori,4 Maria Rhein,1 Laura L. Sutherland,1 Richard Scearce,1
and Barton F. Haynes1
Duke Human Vaccine Institute and Department of Medicine1 and Department of Surgery,4 Duke University School of
Medicine, Durham, North Carolina 27710; Vaccine Research Center, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, Maryland 208922; and Department of Pediatrics, Tulane
University School of Medicine, New Orleans, Louisiana 701123
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Received 29 September 2003/Accepted 16 January 2004
One strategy for the generation of broadly reactive neutralizing antibodies (NA) against human immuno-
deficiency virus type 1 (HIV-1) primary isolates is to use immunogens that have constrained HIV-1 envelope
gp120 conformations reflective of triggered envelope on the surface of virions. A major change in gp120
following binding to CD4 is the enhanced exposure of the CCR5 binding site. One inducer of CCR5 binding site
epitopes on gp120 is the human anti-gp120 monoclonal antibody, A32. We have made cross-linked A32-
rgp12089.6 and A32-rgp120BaL complexes and have compared their immunogenicities to those of uncomplexed
recombinant gp120BaL (rgp120BaL) and rgp12089.6. A32-rgp12089.6 and A32-rgp120BaL complexes had stable
induced CCR5 binding site expression compared to that of uncomplexed rgp120s. However, the A32-rgp120
complexes had similar capacities in guinea pigs for induction of NA against HIV-1 primary isolates versus that
of rgp120 alone. A32-rgp12089.6 induced antibodies that neutralized 6 out of 11 HIV-1 isolates, while rgp12089.6
alone induced antibodies that neutralized 4 out of 11 HIV-1 isolates. A32-rgp120BaL complexes induced
antibodies that neutralized 4 out of 14 HIV-1 isolates while, surprisingly, non-cross-linked rgp120BaL induced
antibodies that neutralized 9 out of 14 (64%) HIV-1 isolates. Thus, stable enhanced expression of the core-
ceptor binding site on constrained gp120 is not sufficient for inducing broadly neutralizing anti-HIV-1 NA.
Moreover, the ability of HIV-1 rgp120BaL to induce antibodies that neutralized ⬃60% of subtype B HIV-1
isolates warrants consideration of using HIV-1 BaL as a starting point for immunogen design for subtype B
HIV-1 experimental immunogens.
The design of immunogens that will neutralize a broad spec- does not bind at the CD4 binding site (26). Thus, MAb A32 has
trum of human immunodeficiency virus type 1 (HIV-1) pri- a potential advantage over CD4 in a constrained Env complex
mary isolates is a high priority for development of a practical in that A32-rgp120 complexes have exposed CD4 binding sites.
HIV-1 vaccine. Following binding of virion gp120 to cellular Here we show that both A32-rgp12089.6 and A32-rgp120BaL
CD4, the HIV-1 envelope undergoes conformational changes complexes are immunogenic and induce NA against HIV-1
that result in exposure of the coreceptor binding site leading to primary isolates. However, stable expression of the CCR5
virion-host cell fusion (1, 24). binding site on gp120 was not sufficient for induction of broad
One potential strategy for inducing broadly reactive neutral- NA, as A32-rgp120 complexes did not show marked enhanced
izing antibodies (NA) is to construct immunogens that are immunogenicity for NA induction over uncomplexed rgp120s.
constrained and reflect wild-type fusion intermediate Env Surprisingly, we found that monomeric recombinant gp120BaL
forms, with the hope of stably exposing conserved immuno- (rgp120BaL) was the best immunogen tested and induced NA
genic epitopes that otherwise would not be readily available for to 64% (9 out of 14) of HIV-1 isolates tested.
antibody induction. An alternative strategy for selection of Env
immunogens is to select the best envelopes from among many
MATERIALS AND METHODS
screened for their ability to induce antibodies that broadly
HIV-1 gp120 proteins. Recombinant vaccinia viruses (rVVs) that express
neutralize HIV-1 primary isolates.
HIV-1 (subtype B) 89.6 gp120 (VBD-2) and HIV-1IIIB (VPE-50) were obtained
In this work we describe the immunogenicity of recombinant from Pat Earl and Bernard Moss (National Institutes of Health [NIH], Bethesda,
HIV-1 gp120s complexed with the CD4 mimic, monoclonal Md.) (19). rVV that expresses group M consensus (CON6) rgp120 (11) was
antibody (MAb) A32. Like CD4, MAb A32 induces expression generated as described previously (19). Briefly, a DNA fragment encoding CON6
of the CCR5 binding site on rgp120, but unlike CD4, MAb A32 gp120 was produced by introducing stop codons after the gp120 cleavage site
(REKR) by PCR and was cloned into a transfer vector, pSC65 vector (from
Bernard Moss) at SalI and KpnI restriction enzyme sites (3). BSC-1 cells were
seeded at 2 ⫻ 105 in each well in a 6-well plate and were infected with wild-type
* Corresponding author. Mailing address: Box 3258 Human Vaccine vaccinia virus (WR) at a multiplicity of infection of 0.1 PFU/cell, and 2 h after
Institute, Duke University Medical Center, Durham, NC 27710. infection pSC65-derived plasmids containing CON6 env genes were transfected
Phone: (919) 684-5858. Fax: (919) 681-8992. E-mail: liao0001@mc into the VV-infected cells by using Lipofectamine 2000 based on the protocol
.duke.edu. recommended by the manufacturer (Invitrogen, Carlsbad, Calif.). rVV that ex-
5270VOL. 78, 2004 CONSTRAINED MAb A32-HIV Env gp120 COMPLEXES 5271
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FIG. 1. Reactivity of HIV-1 MAb A32 with HIV-1 rgp12089.6 and rgp120BaL proteins in Biacore assays. MAb A32 was covalently immobilized
to a CM5 sensor chip (Biacore), and rgp12089.6 (A) and rgp120BaL (B) proteins were injected over each surface. Overlay of curves of Biacore
analysis shows that anti-HIV MAb A32 (solid line) reacted with both rgp12089.6 (A) and rgp120BaL (B) proteins. Curves in dotted lines represent
background binding to an irrelevant IgG. To determine induction of 17b MAb binding to rgp12089.6 (C) and rgp120BaL (D), HIV-1 rgp12089.6
(C) and HIV-1 rgp120BaL (D) proteins were flowed over 17b MAb, which was immobilized on a Biacore chip and was tested for the ability to bind
HIV-1 MAb 17b in the presence or absence of MAb A32 whole IgG or Fab fragment. The x axis shows the duration of time (in seconds), and the
y axis shows the response unit of MAb 17b binding in the Biacore assay. HIV-1 rgp12089.6 constitutively bound MAb 17b (C), indicating the
availability of the CCR5 binding site, while rgp120BaL did not bind to MAb 17b (D). Both whole IgG and Fab fragment of MAb A32 markedly
upregulated (for rgp12089.6) (C) or induced (for rgp120BaL) (D) MAb 17b binding.
presses the CON6 env gene was selected and confirmed by PCR and sequencing under reducing and nonreducing conditions by Coomassie staining, Western blot
analysis as described previously (19). HIV-1 rgp12089.6, HIV-1 rgp120IIIB, and analysis, and Biacore binding assay. Western blots were developed with a satu-
CON6 rgp120 were expressed in 293T cells infected with VBD-2, VPE-50, and rating concentration of HIV-1 envelope protein-specific MAb T8 that reacts with
CON6 gp120 rVV, respectively. Serum-free tissue culture supernatants of 293T most subtype B gp120s at the C1 region (gift of P. Earl) to detect rgp120, and
cells were harvested 3 days after infection with rVVs as a source for purification anti-human immunoglobulin (Ig) (1:4,000; Sigma, St. Louis, Mo.) to detect MAb
of rgp120 proteins. rgp120 proteins in the supernatants were all purified by A32. Surface plasmon resonance binding assays were performed on a Biacore
agarose Galanthus nivalis lectin chromatography (Vector Labs, Burlingame, 3000 (Uppsala, Sweden). Monoclonal antibodies (17b, T8 A32) were immobi-
Calif.) and were stored at ⫺70°C until use. Purified rgp120 proteins were quality lized on a CM5 sensor chip by using standard amine coupling chemistry.
controlled with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- Immunization regimens and adjuvants. Outbred Hartley guinea pigs were
PAGE) followed by Coomassie staining and Western blot. rgp120 proteins of immunized with either 100 g of rgp120 or 200 g of rgp120-A32 complexes in
HIV-1 isolates of BaL (subtype B), 96ZM651 (subtype C), and JRFL (subtype B) either CFA/IFA (Sigma), Ribi-CWS Adjuvant (Corixa-Sigma, St. Louis, Mo.), or
were obtained from QBI Inc., Bethesda, Md., through the NIH AIDS Reagent RC529-SE⫹ mutant cholera toxin (CT) subcutaneously (the gifts of John El-
Repository Program. dridge; Wyeth, Pearl River, N.Y.) every 3 weeks for five immunizations. Serum
Cross-linking of MAb A32 and rgp120. HIV-1 rgp2089.6 or rgp120BaL was samples were collected 10 days after each immunization and were stored at
chemically cross-linked to MAb A32 by using the water soluble N-hydroxysuc- ⫺30°C until use in enzyme-linked immunosorbent assay (ELISA) and neutral-
cinimide-ester dithiobis-sulfosuccinimidylporpionate (DTSSP) (Pierce, Rock- ization assays.
ford, Ill.) based on the protocol recommended by the manufacturer. Briefly, Neutralization assays. HIV-1 isolates (ADA, JRCSF, JRFL, BaL, SF162, and
MAb A32 was mixed with a final concentration of 1 mg of rgp120/ml and was 89.6) were obtained from the NIH AIDS Research and Reference Reagent
incubated at 4°C for 2 h in a 2.25-fold molar excess over MAb A32 to ensure Program (Division of AIDS, National Institute of Allergy and Infectious Dis-
saturation of the bivalent MAb and to have only excess rgp120 following cross- eases). The viruses were expanded by two or three cycles of growth on phyto-
linking. DTSSP was added to the mixture at either 100-fold molar excess for hemagglutinin and interleukin-2-stimulated peripheral blood mononuclear cell
rgp12089.6 or 200-fold molar excess for rgp120BaL at 0°C for 2 h. The reaction was (PBMC). Viruses BL01 and BR07 were provided by Dana Gabuzda of the
stopped by adding 1 M Tris at a final concentration of 50 mM and was further Dana-Farber Cancer Institute. Both are chimeric infectious molecular clones of
incubated at 20°C for 30 min. NL4-3 that contain the full-length env genes from primary HIV-1 isolates (20).
Cross-linked A32-rgp120 complexes were separated from uncomplexed gp120 After initial plasmid transfection of 293 cells, these viruses were expanded in
proteins by size exclusion chromatography over a HR200 Superdex column and PBMC as described above.
by using an AKTA fast protein liquid chromatography (FPLC) system (Uppsala, Serum NA levels were measured either by flow cytometic enumeration of p24
Sweden). Fractions were analyzed on 4 to 20% Tris-Glycine gels (Invitrogen) antigen-positive cells following a single round of infection (16) or by using a5272 LIAO ET AL. J. VIROL.
assay) of AT-2-inactivated HIV-ADA or ADA (from Larry Arthur) for 1 h at
37°C. After 1 h of incubation the mixtures of serum and virus were incubated
with Sup T1 cells (100,000 cells per well of 96-well half-area microtiter plates)
overnight in a 37°C, 5% CO2 incubator. The presence of characteristic syncytia
was evaluated by inverted phase-contrast microscopy 16 h after virus addition.
Numbers of syncytia were counted per view at 100⫻ magnification under a
phase-contrast light microscope. Serum titer was determined as a serum sample
at a given dilution giving ⱖ90% inhibition of syncytium formation compared to
that of prebleed sera.
Anti-gp120 serum antibody assay. Binding antibody titers against a panel of
HIV-1 rgp120 proteins were measured in a standard ELISA. HIV-1 rgp120s
from various HIV-1 isolates, including subtype B isolates of 89.6, JRFL, IIIB,
BaL, subtype C isolates of 96ZM651, and CON6. HIV-1 rgp120 proteins were all
used at a concentration of 2 g/ml (200 ng/well) to coat 96-well ELISA microtiter
plates. Antibody endpoint binding titers were determined as the reciprocal of the
highest dilution of the serum assayed against corresponding recombinant HIV-1
envelope proteins, giving optical density readings of experiment versus control of
ⱖ3.0.
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RESULTS
Enhanced binding of MAb 17b to A32-rgp120 complexes.
MAb A32 broadly reacts with a wide spectrum of HIV-1 pri-
mary isolates from different subtypes (26). By using surface
plasmon resonance assays we found MAb A32 reacted with
both rgp2089.6 and rgp120BaL (Fig. 1A and B). HIV-1 MAb 17b
constitutively bound to rgp2089.6 but was nonreactive with
rgp120BaL (Fig. 1C and D). Incubation of MAb A32 whole IgG
FIG. 2. FPLC elution pattern of the cross-linked A32-gp120 com-
as well as Fab fragments with HIV-1 rgp120 proteins induced
plexes on a Superdex H200 column. MAb A32-rgp12089.6 (A) and markedly enhanced MAb17b binding to rgp2089.6 (Fig. 1C)
A32-rgp120BaL (B) complexes were made as described in Materials and induced de novo MAb 17b binding to rgp120BaL (Fig. 1D).
and Methods and fractionated on a Superdex HR200 column. Frac- Production of A32-rgp120 complexes with the CCR5 binding
tions were collected and assayed for the ability to bind MAb 17b, site stably exposed. The presence of stably exposed or induced
immobilized on a Biacore CM5 sensor chip. Circles represent binding
levels of each fraction to immobilized 17b MAb, while the UV trace CCR5 binding sites on constrained HIV-1 envelope proteins
from the FPLC run is shown with a solid line. Also shown is that the indicates induction of conformational changes in gp120 follow-
enhanced level of MAb 17b binding was obtained in fractions under ing CD4 or MAb A32 binding (1, 13, 23, 26). While the CCR5
peak 1 (molecular size of about 440 kDa) that contained the cross- binding site itself might not be a target for broadly reactive NA
linked rgp12089.6 (A) and rgp120BaL (B). In contrast, peak 2 containing
excess gp120 did not show upregulated MAb 17b binding.
(8), in this study the conformational exposure of the 17b bind-
ing site was monitored as an indicator of A32-induced confor-
mational changes in rgp120. Our postulate was that A32-trig-
gered rgp120 might possess conserved conformational epitopes
syncytium inhibition assay with aldrithiol-2 (AT-2)-inactivated virions (21) or a against which broadly reactive NA could be made. Thus, we
luciferase-based multiple-round HIV-1 infection assay (2). The single-round flow
cytometric assay for intracellular p24 antigen was performed as described pre-
produced A32-HIV-1 rgp120 complexes by cross-linking MAb
viously (16). In the luciferase-based assay, NA were measured as a function of a A32 and either gp12089.6 or rgp120BaL with DTSSP. To purify
reduction in luciferase activity in 5.25.EGFP.Luc.M7 cells kindly provided by A32-rgp120 complexes and to remove uncross-linked rgp120,
Nathaniel R. Landau (Salk Institute, La Jolla, Calif.) (2). Five hundred 50% A32-gp12089.6 and A32-rgp120BaL stable complexes were pu-
tissue culture infectious doses (TCID50) of cell-free virus were incubated with
rified on Superdex HR200 gel filtration columns. Figure 2
the indicated serum dilutions in a volume 150 l (1 h at 37°C) in triplicate in
96-well flat-bottom culture plates. The 5.25.EGFP.Luc.M7 cells were suspended shows that the higher molecular sizes of A32-rgp2089.6 (Fig.
at a density of 5 ⫻ 105/ml in media containing diethylaminoethyl dextran (10 2A) and A32-rgp120BaL (Fig. 2B) complexes were eluted first
g/ml). Cells (100 l) were added until 10% of cells in control wells (no test and could be separated from uncross-linked rgp120. We next
serum sample) were positive for green fluorescent protein expression by fluo- examined each fraction off the Superdex HR200 FPLC column
rescence microscopy. At this time the cells were concentrated twofold by remov-
ing one-half of the volume of medium. A 50-l suspension of cells was trans-
for constitutive binding activity to MAb 17b immobilized on a
ferred to 96-well white solid plates (Costar, Cambridge, Mass.) for measurement Biacore chip. We found that all upregulated binding to 17b was
of luciferase activity by using Bright-Glo substrate (Promega, Madison, Wis.) on indeed in the A32-rgp120 complex in peak 1 (Fig. 2A) fractions
a Wallac 1420 Multilabel Counter (PerkinElmer Life Sciences, Boston, Mass.). containing A32-gp12089.6 complexes. Similarly, analysis of the
Neutralization titers in the luciferase assays were those where ⬎50% virus
A32-rgp120BaL complexes showed stable induced MAb 17b
infection was inhibited and were considered positive if the postimmune bleed
titer minus the preimmune bleed titer was ⬎30 and the postbleed titer was three binding (Fig. 2B).
times higher than the prebleed titer. For analysis of breadth, those isolates that Figure 3 shows SDS-PAGE gels of unpurified A32,
were neutralized by at least 2 sera per group were considered positive. rgp2089.6, and A32-rgp12089.6 complexes both before and after
The syncytium inhibition fusion-from-without assay utilized HIV-1 AT-2-in- separation on a Superdex HR200 FPLC column. MAb A32
activated virions from HIV-1 subtype B strains ADA and AD8 (the gift of Larry
Arthur and Jeffrey Lifson, Frederick Research Cancer Facility, Md.). Twofold
sized at ⬃210,000 Da while rgp12089.6 was 120,000 Da. Unpu-
serially diluted immune and control sera starting at 1:10 dilution were incubated rified A32-rgp12089.6 complexes contained higher-molecular-
with optimal dilution (1:80, which results in approximately 80 syncytia in the size complexes as well as excess uncomplexed rgp120. Peak 2 inVOL. 78, 2004 CONSTRAINED MAb A32-HIV Env gp120 COMPLEXES 5273
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FIG. 3. SDS-PAGE and Western blot analysis of unfractionated and FPLC-fractionated A32-rgp120 complexes. MAb A32-rgp12089.6 com-
plexes were made as described in Materials and Methods, fractionated on a Superdex HR200 FPLC column (bottom panel), analyzed, and
compared to unfractionated rgp120, A32, and A32-rgp120 by Coomassie staining (A) and Western blot (B) of the reducing (middle frame of panel
A) and nonreducing (top frame of panel A) SDS-PAGE. In Western blotting run under nonreducing conditions, HIV-1 envelope-specific MAb
T8 was used to detect rgp12089.6 in the A32-rgp12089.6 complexes (top frame of panel B), and anti-human Ig was used to detect human MAb A32
(middle frame of panel B). Samples in the individual lanes are corresponding the fractions off the Superdex H200 FPLC column (bottom frames
of panels A and B). The analysis demonstrates that complexes made with one gp120 or two gp120s bound to MAb A32 indeed contained both MAb
A32 and rgp12089.6.
the chromatogram below contained free rgp120 (fraction lane Ability of antisera against A32-rgp120 complexes to react
23, 24, and 25), while A32-rgp12089.6 complexes eluted in peak with a panel of HIV-1 rgp120s. To determine if cross-linking
1 (fractions 18 to 21). The weak lower-molecular-size band in 89.6 or BaL rgp120s prevented induction of anti-gp120 anti-
fraction 21 reflected complexes comprised of one rgp120 and bodies, we screened anti-A32-rgp120 complex antisera for re-
one A32 molecule, while the higher-molecular-size band in activity with a panel of four subtype B rgp120s (89.6, JRFL,
fractions 18 to 20 reflected saturated A32 MAb with two bound IIIB, and BaL), a group M consensus Env rgp120 (CON6), and
rgp120s. Under reducing conditions (the middle panel) the a subtype C rgp120 (96ZM651). We found that both 89.6 and
complexes in fractions 18 to 21 all contained rgp120 as well as BaL rgp120 complexed with A32-induced anti-gp120 antibod-
55,000 Da of Ig heavy chains and 29,000 Da of Ig light chains. ies equally well, with both types of antisera reacting with each
In Fig. 3B, Western blot with an anti-human Ig (middle panel) rgp120 (Fig. 4).
and anti-gp120 mouse MAb T8 (lower panel) confirmed that NA induced by A32-rgp120 complexes compared to that of
the complexes in peak 1 contained both rgp120 and MAb A32. rgp120 alone. Next we compared the ability of A32-rgp12089.6
Thus, both A32-rgp12089.6 and rgp120BaL complexes had sta- (Table 1) and A32-rgp120BaL (Table 2) complexes to
ble expression of the CCR5 binding site, and these complexes rgp12089.6 (Table 1) and rgp120BaL (Table 2) alone for their
(lanes 18 to 21 in Fig. 3) were used in a series of immunizations ability to induce anti-HIV-1 neutralizing antibodies. While
in guinea pigs. A32-rgp120 complexes induced antibodies that neutralized5274 LIAO ET AL. J. VIROL.
rgp120 alone and that induced by A32-rgp120 complexes.
While A32-rgp12089.6 complexes induced antibodies that neu-
tralized two additional HIV-1 isolates more than A32-
rgp12089.6 alone (IIIB, JRCSF), the A32-rgp120 complexes did
not induce antibodies that broadly neutralized five of the more
difficult-to-neutralize HIV-strains (US-1, US717, BL01, 6101,
and BG1168).
To determine if A32-rgp120 complexes induced antibodies
that inhibited HIV-1-induced fusion, we tested the sera listed
in Tables 1 and 2 for the ability to inhibit inactivated HIV-1
induced syncytium from without by using AT-2-inactivated viri-
ons ADA and AD8 (Table 3). We found most postimmune
sera had syncytium inhibiting activity, with the anti-rgp120 sera
titers as high or higher than those for the anti-A32-rgp120
complex antisera.
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FIG. 4. Reactivity of guinea pig anti-HIV-1 sera to a panel of Because the rgp120s performed as well as the A32-rgp120
HIV-1 rgp120. To determine reactivity of guinea pig postimmune sera complexes, we next determined the ability of antisera against 89.6
to HIV-1 gp120 envelope proteins in ELISA, a panels of HIV-1 rgp120 and BaL rgp120s to neutralize seven subtype B HIV-1 isolates in
proteins (200 ng/well) as indicated in the X axis were coated in 96-well a luciferase-based multiple round infection NA assay (Table 4).
microtiter plates and tested for the reactivity of sera from guinea pigs
immunized with either A32-rgp12089.6 or rgp120BaL. The endpoint Four isolates were the same as those tested in the single-round
ELISA titers of guinea pig sera are determined as described in Mate- p24 assay (BaL, BG1168, 6101, SF162), and three were different
rial and Methods and are shown in the Y axis. (BX08, SS1196 and QH0692). While rgp12089.6 antisera only
strongly neutralized BX08 (2 out of 3 were serum positive),
rgp120BaL antisera strongly neutralized SF162, BX08, SS1196,
multiple HIV primary isolates (A32-rgp12089.6, 6 out of 11 and QH0692 in addition to BaL and SF162.
isolates neutralized; A32-rgp120BaL, 4 out of 11 isolates neu- Taken together with the data in Tables 1 and 2, rgp12089.6
tralized), there were no profound differences between the neutralized 4 out of 14 HIV-1 isolates (29%) while rgp120BaL
number of HIV isolates neutralized by antisera induced by antisera neutralized 9 out of 14 HIV-1 isolates (64%).
TABLE 1. Ability of MAb A32 complexed with HIV 89.6 gp120 versus 89.6 gp120 alone to induce antibodies that neutralize HIV
primary isolates
Guinea pig no. or % Neutralization against HIV-1 isolateb
reference control Immunogen
antibodya IIIB BaL 89.6 SF162 JRCSF US1 US717 BL01 BR07 6101 BG1168
Animal
504 A32-gp12089.6 complex* 53 52 99 76 73 51 37 29 99 0 0
505 A32-gp12089.6 complex* 29 39 96 65 54 13 10 22 97 8 0
506 A32-gp12089.6 complex* 53 19 94 80 52 40 0 31 90 0 0
561 A32-gp12089.6 complex# 11 53 97 87 50 28 18 4 95 0 12
562 A32-gp12089.6 complex# 90 27 95 89 53 6 13 0 91 0 0
563 A32-gp12089.6 complex# 8 48 91 80 42 29 0 3 60 0 1
507 rgp12089.6* 21 8 99 84 47 0 2 12 99 22 4
508 rgp12089.6* 32 32 97 81 73 44 3 36 97 41 0
509 rgp12089.6* 32 11 96 73 48 3 9 50 90 42 5
568 rgp12089.6# 59 5 98 81 8 9 9 10 72 39 20
510 A32 MAb* 0 8 9 2 24 48 14 32 13 38 0
511 A32 MAb* 0 0 15 16 28 30 21 7 27 40 9
512 A32 MAb* 0 27 24 48 45 31 36 40 54 11 21
565 A32 MAb# 14 2 0 12 10 7 15 0 0 10 15
566 A32 MAb# 11 31 3 10 11 14 7 19 0 35 2
Antibody control
HIV Ig (10,000) 100.2 99.5 99.9 99.9 99.2 97.8 99.7 100.1 97.9 99.3 100.2
HIV Ig (1,000) 92.9 80.1 65.2 94.6 75.2 59.7 45.8 91.3 53.7 67.2 61.4
2F5 (50) 97.1 81.3 98.9 96.5 97.1 95.8 91.9 99.2 94.3 37.6 95.0
2F5 (5) 73.4 47.5 82.0 65.8 65.3 66.2 52.6 74.3 66.6 29.9 59.0
2G12 (50) 86.4 82.2 76.4 52.7 61.0 52.5 ⫺10.9 38.0 46.6 86.5 ⫺6.9
2G12 (5) 71.8 28.2 56.2 29.4 34.4 33.7 ⫺17.7 27.5 16.5 25.5 ⫺24.8
a
*, adjuvant CFA/IFA; #, adjuvant RC529 plus mCT. The numbers in parentheses following antibody designations are the amounts of virus isolate used in
micrograms per milliliter.
b
Neutralization assays were done by flow cytometric enumeration of p24 antigen-positive cells after a single round of infection of CD8-depleted PBMC. Percent
neutralization values were determined by comparing results for immune sera to the results for corresponding preimmune sera at a 1:5 dilution. Values that are ⱖ50% are
positive.VOL. 78, 2004 CONSTRAINED MAb A32-HIV Env gp120 COMPLEXES 5275
TABLE 2. Ability of MAb A32 complexed with HIV BaL gp120 versus BaL gp120 alone to induce antibodies that neutralize HIV
primary isolates
Guinea pig no. or % Neutralization against HIV-1 isolateb
reference control Immunogen
antibodya IIIB BaL 89.6 SF162 JRCSF US1 US717 BL01 BR07 6101 BG1168
Animal
513 A32-gp120BaL complex† 60 78 61 88 86 42 35 0 46 21 11
514 A32-gp120BaL complex† 29 88 53 70 46 0 2 0 61 8 7
577 A32-gp120BaL complex† 45 95 22 72 64 31 8 19 0 35 11
573 rgp120BaL† 72 82 90 80 88 49 6 29 81 24 0
574 rgp120BaL† 39 100 67 82 77 41 25 5 75 9 0
578 rgp120BaL† 66 99 68 85 64 24 21 32 30 0 6
510 A32 MAb* 0 8 9 2 24 48 14 32 13 38 0
511 A32 MAb* 0 0 15 16 28 30 21 7 27 40 9
512 A32 MAb* 0 27 24 48 45 31 36 40 54 11 21
565 A32 MAb# 14 2 0 12 10 NT NT 0 0 10 NT
566 A32 MAb# 0 8 9 2 24 48 14 32 13 38 0
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Antibody control
HIV Ig (10,000) 100.2 99.5 99.9 99.9 99.2 97.8 99.7 100.1 97.9 99.3 100.2
HIV Ig (1,000) 92.9 80.1 65.2 94.6 75.2 59.7 45.8 91.3 53.7 67.2 61.4
2F5 (50) 97.1 81.3 98.9 96.5 97.1 95.8 91.9 99.2 94.3 37.6 95.0
2F5 (5) 73.4 47.5 82.0 65.8 65.3 66.2 52.6 74.3 66.6 29.9 59.0
2G12 (50) 86.4 82.2 76.4 52.7 61.0 52.5 ⫺10.9 38.0 46.6 86.5 ⫺6.9
2G12 (5) 71.8 28.2 56.2 29.4 34.4 33.7 ⫺17.7 27.5 16.5 25.5 ⫺24.8
a
*, adjuvant CFA/IFA; #, adjuvant RC529 plus mCT; †, adjuvant RiBi/CWS. The numbers in parentheses following antibody designations are the amounts of virus
isolate used in micrograms per milliliter.
b
Neutralization assays were done by flow cytometric enumeration of p24 antigen-positive cells after a single round of infection of CD8-depleted PBMC. Percent
neutralization values were determined by comparing results for immune sera to the results for corresponding preimmune sera at a 1:5 dilution. Values that are ⱖ50%
are positive.
Thus, for both the single-round neutralization assays and the induce broadly reactive NA. First, because of a concern that
inhibition of virion-induced fusion assays, rgp120 was equal the oil adjuvants described in Tables 1 and 2 might interfere
(89.6) or superior (BaL) to A32-rgp120 complex induction of with the ability of constrained rgp120 to express important
anti-HIV-1 NA. conformational neutralizing epitopes, we formulated A32-
Evaluation of the effect of adjuvants and DTSSP cross- rgp12089.6 complexes in 10 g of CT as an adjuvant in saline
linking on A32-rgp120 immunogenicity. We next performed and immunized SQ X3, followed by 50 g of lipopolysac-
two additional sets of immunizations to evaluate conditions charide plus 10 g of CT in saline for an additional two
that might affect the ability of A32-rgp120 complexes to boosts. This regimen achieved an endpoint titer of 1:204,800
against rgp12089.6 in both animals tested, an ELISA titer
that is sufficient for demonstrating the presence of NA.
TABLE 3. Comparison of A32-gp120 complexes and rgp120 to Table 5 shows that 1 of 2 animals immunized with A32-
induce antibodies that inhibit syncytia formation by
AT-2-inactivated virions rgp2089.6 complexes in saline plus CT plus LPS (no. 572)
neutralized 5 of 11 HIV-1 isolates while the other serum
90% Syncytia inhibition
titerb for:
(no. 570) neutralized none. The sera from animals immu-
Guinea
pig no.
Immunogen a
nized with rgp12089.6 alone neutralized from 2 to 4 HIV-1
HIV-1 HIV-1
ADA AD8
isolates (Table 4). As for Table 1, there was a suggestion of
more breadth with A32-rgp12089.6 complexes with more
504 A32-gp12089.6 90 270 neutralization in serum 572 of HIV-1 IIIB and BaL than
505 A32-gp12089.6 90 10
506 A32-gp12089.6 ⬍10 ⬍10
with rgp12089.6 immune sera. However, the A32-rgp120
561 A32-gp12089.6 10 90 complex sera with saline adjuvants again did not neutralize
562 A32-gp12089.6 30 270 HIV-1 isolates B61168, 6101, BL01, US17, or US717.
563 A32-gp12089.6 30 30 The second set of control immunizations performed was
507 rgp12089.6 90 90 with rgp120BaL treated with DTSSP in the absence of A32
508 rgp12089.6 270 90
509 rgp12089.6 ⬍10 ⬍10 MAb. In contrast to when A32 was used in the complex, we
568 rgp12089.6 30 270 found that postimmune sera from three animals (H12, H13,
513 A32-gp120BaL 270 270 H14) immunized four times with DTSSP-treated rgp120BaL
514 A32-gp120BaL 30 30 neutralized 0 of 11 of the HIV-1 isolates listed in Table 4
577 A32-gp120BaL 90 30
573 rgp120BaL ⬎810 ⬎810 (data not shown). Taken together, these controls demon-
574 rgp120BaL ⬎810 ⬎810 strated that (i) the oil adjuvants described in Tables 1 and 2
578 rgp120BaL 270 270 did not prevent expression or recognition of conserved
a
Adjuvants used were the same as those described in Tables 1 and 2. epitopes exposed on the surface of A32-rgp120 and
b
Reciprocal dilution at which ⬎90% of syncytia are inhibited. epitopes, and (ii) DTSSP treatment of rgp120BaL in the5276 LIAO ET AL. J. VIROL.
TABLE 4. Comparison of HIV-1 gp120 BaL and gp120 89.6 to induce antibodies that neutralize HIV-1 primary isolates
Guinea Neutralizing antibody titer of guinea pig sera against HIV-1 primary isolatea
Immunogen
pig no. BaL BX08 BG1168 6101 SF162 SS1196 QH0692
573 BaL gp120 >540 >540 ⬍20 ⬍20 1,332 317 151
574 BaL gp120 >540 >540 ⬍20 ⬍20 1,068 415 157
578 BaL gp120 >540 540 ⬍20 ⬍20 432 71 ⬍20
507 89.6 gp120 ⬍20 128 ⬍20 ⬍20 >540 ⬍20 ⬍20
508 89.6 gp120 ⬍20 215 ⬍20 ⬍20 4,591 47 38
509 89.6 gp120 ⬍20 ⬍20 ⬍20 ⬍20 374 ⬍20 22
568 89.6 gp120 ⬍20 ⬍20 ⬍20 ⬍20 151 ⬍20 ⬍20
a
Assays were performed in the M7-luciferase-based assay, and titers are reciprocal dilutions of guinea pig serum at which 50% neutralization occurs. Boldface
numbers are those titers that were ⬎30 and postimmune sera that were three or more times greater than those of prebleed serum titers.
absence of MAb A32 eliminated the immunogenicity of reasons: (i) MAb A32 reacts with many HIV-1 isolates of
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rgp120 neutralizing determinants. Thus, the immunogenic- multiple subtypes, (ii) MAb A32 is as potent an inducer of the
ity of rgp120 in A32 complexes was not due to DTSSP CD4i coreceptor binding site as is sCD4, and (iii) MAb A32
treatment per se. does not bind to the CD4BS, thus leaving it open for induction
of anti-CD4BS antibodies (26).
DISCUSSION While A32-rgp120 complexes with rgp12089.6 induced
slightly more neutralizing breadth than rgp12089.6 alone, com-
In this study we have produced A32-rgp120 complexes with parative studies with A32-rgp120BaL showed that A32-
stably expressed coreceptor binding sites and have compared rgp120BaL complexes were less immunogenic for broadly reac-
these complexes as immunogens with uncomplexed rgp120. tive NA than uncomplexed rgp120BaL (Tables 2 and 5). Thus,
Our study is important in that we determined (i) that stable our carefully characterized A32-rgp120 complexes provide the
enhanced expression of the coreceptor binding site is not suf-
first data demonstrating that exposure of the coreceptor bind-
ficient for induction of broadly reactive NA in the absence of
ing site alone is not sufficient for induction of broadly reactive
CD4, (ii) oil adjuvant formulation did not prevent A32-rgp120
NA. Immunofluorescence studies on live HIV-infected cells
broadly neutralizing antigen expression, and (iii) rgp120BaL
have shown limited accessibility of the coreceptor binding site
alone was the best Env immunogen tested.
during fusion (8). In contrast, the fusion protein CD4-17b Fab
Fouts et al. have reported that cross-linked CD4-rgp120IIIB
is a potent bivalent inhibitor of most HIV-1 primary isolates
and CD4-rgp140IIIB complexes, unlike uncross-linked rgp120
or rgp140, induced antibodies that neutralized a spectrum of (7). Clearly envelope constructs more native than A32-rgp120
HIV-1 primary isolates from multiple HIV-1 subtypes (9). Sol- monomers, such as A32-bound gp140 or gp160 trimers, need to
uble CD4 in cross-linked CD4-rgp120 complexes could be im- be tested as immunogens.
portant as a component for the ability to induce cross-reactive DeVico et al. have shown that expression of CD4-induced
NA in multiple ways. Human CD4 induces anti-CD4 antibod- neutralizing determinants defined by MAbs raised against
ies that themselves neutralize HIV-1. Indeed, absorption of CD4-rgp120 complexes vary in their expression on native
anti-CD4 antibodies removed some but not all of the cross- gp120s, being constitutively expressed on some gp120s and less
subtype neutralizing activity induced by CD4-rgp120 com- so on others (5). In this regard, rgp12089.6 had constitutive
plexes (5, 9). Second, CD4, when complexed to rgp120, can be low-level expression of MAb 17b (7) binding that increased
part of a new neutralizing determinant in the complex and/or after MAb A32 binding while rgp120BaL had no constitutive
induce new neutralizing determinants on rgp120 following in- MAb 17b binding. However, rgp120BaL induced antibodies
duction of conformational changes in rgp120 by CD4 (5, 6, 9). that neutralized 64% (9 of 14) of subtype B isolates tested
We chose to study MAb A32-rgp120 complexes for several while rgp12089.6 was less effective, which could be due to con-
TABLE 5. Ability of MAb A32 complexed with rgp12089.6 versus rgp12089.6 alone formulated in aqueous adjuvants to induce antibodies that
neutralize HIV-1 primary isolates
Guinea % Neutralization against HIV-1 isolatea
Immunogen
pig No. IIIB BAL 89.6 SF162 JRCSF US1 US717 BL01 BR07 6101 BG1168
570 A32-gp12089.6 complex 14 4 48 42 49 0 4 0 0 0 3
572 A32-gp12089.6 complex 50 64 92 80 54 32 31 41 57 5 5
558 rgp12089.6 28 18 98 73 55 24 17 48 87 47 23
559 rgp12089.6 0 22 100 85 34 16 0 9 72 0 0
560 rgp12089.6 0 0 100 82 16 12 0 12 19 0 11
555 A32 MAb 2 19 13 17 12 7 0 29 0 11 27
556 A32 MAb 11 5 18 16 7 17 0 0 15 17 15
557 A32 MAb 0 1 0 3 10 7 0 34 0 0 0
a
% Neutralization values are based on comparison with corresponding preimmune sera at 1:5 dilution. Values that are ⱖ50% are considered positive (boldface).VOL. 78, 2004 CONSTRAINED MAb A32-HIV Env gp120 COMPLEXES 5277
stitutive expression of as-yet undefined conserved neutralizing neutralized by anti-rgp120BaL sera were the isolates known to
determinants on rgp120BaL. These latter data give credence to be easily neutralized (e.g., IIIB, BaL, SF162, 89.6), while the
the strategy of testing multiple HIV-1 primary isolate Envs to more difficult to neutralize HIV isolates (e.g., BG11681, 6101,
select a best Env or best polyvalent Envs for induction of US1) were not neutralized. Thus, rgp120BaL in and of itself is
broadly reactive NA. not likely to solve the HIV-1 vaccine NA immunogen problem,
Generally, most analyses of immune sera from animals and but as a present best Env it can serve as a starting point for
humans have shown little neutralizing activity for HIV-1 pri- design of more native Env immunogens. These strategies in-
mary isolates (13, 14, 17, 18, 22, 24, 25). However, recent clude formulation of trimeric rgp140BaL immunogens and pro-
analysis of immune sera raised against either polyvalent gp120 duction of constrained gp140 trimer complexes. In this regard,
(4), gp120 subunit peptides (15), or oligomeric gp160 (25) have Fouts et al. have described the production of a single-chain
demonstrated the ability to induce antibodies that neutralized polypeptide analogue of the CD4-rgp120 complex utilizing the
select HIV-1 primary isolates. Clearly, the majority of HIV-1 gene for rgp120BaL (10).
primary isolates neutralized by anti-gp120 immune sera in our Taken together, our data suggest that HIV-1 BaL rgp120 is
study are more easily neutralizable than others, and the a promising R5 envelope that can serve as a starting point for
breadth of neutralization elicited by the best immunogen, development of an HIV-1 immunogen for induction of broadly
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rgp120BaL, is not sufficient to anticipate widespread utility as a reactive NA against subtype B HIV-1 primary isolates.
vaccine. It is important to note that AT-2-inactivated HIV-1
virions (21) have been grown in vitro in T-cell lines and are ACKNOWLEDGMENTS
readily neutralized in syncytium from without inhibition assays. We acknowledge Kim McClammy for expert secretarial assistance.
Thus, the syncytium inhibition assay used in our study is useful We acknowledge the Proteomics Core of Human Immunology Cen-
to demonstrate the presence of inhibiting activities, but the ter grant AI-51445, PO-1 AI-52816. This work supported by AI-15351,
assay appears to be more sensitive than the luciferase-base HIV Team Contract grant no. N01-AI05397, AI-24030, and the NIH,
NIAID, AIDS Research and Reference Reagent Program.
multiple-round infection assay, the single-round infection cy-
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