Neutralizing Recombinant Human Antibodies to a
Conformational V2- and CD4-Binding Site-Sensitive Epitope of
HIV-1 gpl20 Isolated by Using an Epitope-Masking Procedure'
Henrik J. Ditzel,2*t James M. Binley,* John P. Moore,* Joseph Sodroski,§ Nancy Sullivan,'
Lynette S. W. Sawyerjl R. Michael Hendryjl Wei-Ping Yang,' Carlos F. Barbas Ill,' and
Dennis R. Burton2*'
*Departments of Immunology and ¶Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037; 'Department of
Medical Microbiology, Odense University Medical School, Odense, Denmark; *Aaron Diamond AIDS Research Center, New
York University School of Medicine, New York, NY 10016; 'Division of Human Retrovirology, Dana-Farber Cancer Institute,
Boston, MA 021 15; and IlViral and Rickettsial Disease Laboratory, California Department of Health Services, Berkeley, CA 94704
As part of the goal of assembling amixture of neutralizing human mAbs for possible prophylaxis and therapy of HIV-1
disease, we describe a strategy by which neutralizing human Abs to a weakly immunogenic epitope can be accessed.
From a phage display library derived from an asymptomatic HIV-1 seropositive donor, a panel of recombinant Fabs
against the CD4 bindingsite (CD4bs) of gpl20 was retrieved by affinity selection using recombinant gpl20 (strain LAI).
Two Fabs corresponding to the dominant clones were used to mask the CD4bs epitope(s) before repeating
the selection
procedure. Four Fabs were then retrieved that had novel heavy chain sequences. Three recognized a novel epitope
distinct from that recognized by conventional CD4bs Abs and were defined by the following criteria: 1) second V region
(V2 region) dependence indicated by sensitivity to amino acid changes in the V2 loop and by competition with murine
anti-V2 mAbs; 2) CD4bs dependence indicated by sensitivity to amino acid changes usually associated with CD4
binding and by inhibitionof Fab binding to gpl20 soluble
by
CD4; this dependence seemed to arise via conformational
changes rather than by direct binding, as CD4bs Abs enhanced binding of two of the novel Fabs and, in a reversal of
the competition format, the novel Fabs did not inhibit soluble CD4 binding to gp120; and 3) equivalent binding to
glycosylated and deglycosylated gpl20 and significant, although much reduced, binding to denatured gpl20 in contrast with CD4bs Abs, which do not bind to deglycosylated or denatured gpl20. One of the novel Fabs efficiently
neutralized the MN and LA1 strainsof HIV-1. These results indicate the presence of a novel neutralizing conformational
epitope on gpl20 sensitive to the V2 loop and the CD4bs and further highlight the conformational flexibility of gpl20.
The strategy of masking highly immunogenic epitopes with Abs to rescue a broader range of specific Abs from combinatorial libraries should be widely applicable. The journal of Immunology, 1995, 154: 893-906.
B
oth the cellular and humoral immune systems are
involved in the immune response against HIV-1,
the etiologic agent of AIDS (1, 2). Abs against
epitopes on the HIV-1 surface glycoprotein gp120 and, to
Received for publication May 12, 1994. Accepted for publication October20,
1994.
The costs of publication of this article were defrayed in part by the payment
of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
'
This work was supported by National Institutes of Health Grant A133292-02
and byJohnson &Johnson.H.J.D.was supported by the Danish Research Council
and Odense University School of Medicine. ].P.M. was supported by the Aaron
Diamond Foundation and New York University School of Medicine CFAR. J.S.
was supported by National Institutes of Health Grants A124755 and A131 783, by
Cancer Center (CA06516) andCenter for AIDS Research (A128691) Grants to the
Dana-Farber Cancer Institute, and by gifts from the G. Harold and Leila Y. Mathers
Charitable Foundation and from the late William McCarty-Cooper. C.F.B. is a
Scholar of the American Foundation for AIDS Research and a recipient of a
Scholar Award from the Cancer Research Institute.
Copyright 0 1995 by The American Association of Immunologists
lesser extent, the transmembrane glycoprotein gp41 constitute most of the neutralizing humoral response to HIV-1
(3, 4). Such serum Absarecapable
of inhibiting both
HIV-1 cellfree infectivity and cell-to-cell spread.
gp120 is a heavily glycosylated protein with multiple
internal disulfide bonds (5-7). Comparison of divergent
HIV-1 isolates has revealed that the structureof gp120 can
be separated into five relatively conserved (C1 through
C5) and five variableregions (V1 through VS) (7, 8).
Three regions on the gp120 molecule seem to beespecially important for Ab-mediated virus neutralization: the
'Addresscorrespondenceandreprint
requests toDr.
DennisBurtonor
Dr. Henrik Ditzel, Departmentof Immunology, The Scripps Research Institute,
10666 North Torrey Pines Road, La Jolla, CA 92037.
0022.1 767/95/$02.00
894
V33 region and the discontinuous epitope forming the
CD4 binding site (CD4bs) have long been recognized as
epitopes for neutralizing Abs (9-13), and recent data indicate that rodent Abs recognizing the V2 region also mediate virus neutralization (14-18). Whereas the CD4bs is
responsible for the initial binding of the virus to the CD4
molecule on the surface of target cells (19, 20), both the
V2 and the V3 regions are important for the subsequent
virus-cell fusion process, although the exact mechanisms
are unknown (21-27). It has often been considered that the
CD4bs, V2 region, and V3 region compriseseparate
epitopes, although recent reports indicate someinterdependence between distinct domains of the same protein
(17, 28-30).
Given the strain heterogeneity of the HIV-1 virus (31), an
Ab-based strategy for therapy or prophylaxis will likely require Abs targetedto several epitopes. The preparation of
combinatorial libraries from variable heavy and light chain
Ab genes provides an efficient route for the selection of high
affinity human mAbs (32). From such libraries, Abs of interest can be cloned, using Ag-binding as the selection system.
The construction of such Ab libraries on the surface of m13
bacteriophage has been described (33-35), as has their application to the generation of a large range of human mAbs
against viruses, including HIV-1, RSV, HSV-1, HSV-2,
VZV, CMV, and hepatitis B (12, 36-40).
Obtaining Abs with different specificities against a
given Ag from combinatorial libraries can be difficult because certain epitopes may dominate. This would seem to
be the case for the CD4bs of gp120, in that virtually all of
the Abswe obtained from a library derived from an
asymptomatic HIV-1 seropositive donor were directed to
this site and were competitive with one another (12, 36,
37). Abs recognizing the V3 loop of HIV-1 MN were selected from the same library by using a constrained peptide corresponding to the tip of the loop (36, 37).
Here we report on the preparation of an additional library from a long-term asymptomatic HIV-1 seropositive
individual and describe a method by which Abs to minor
epitopes can be obtained. Human monoclonal Fabs to a
novel epitope sensitive to both the V2 region and the
CD4bs of gp120 were generated along with Fabs directed
against the CD4bs. Several of the Fabs to the CD4bs, and
one to the novel epitope, showed potent neutralizing ability for laboratory strains of HIV-1.
Materials and Methods
Lymphocyte RNA preparation and library
construction
RNA was prepared from 5 ml of bone marrow lymphocytes aspirated
from a long-term (>6 years) asymptomatic HIV-1 seropositive donor.
Abbreviations used in this paper: V3 region, third variable region;V2 region,
CD4 bindingsite; SB medium,superbroth
secondvariableregion;CD4bs,
medium; AP, alkalinephosphatase; SATA, N-succinimidyl-S-acetylthioacetale; NPP, nitrophenol substrate.
NEUTRALIZING HUMAN HIV-1 AbsLIBRARIES
FROM PHAGE
After reverse-transcription, the yl-(Fd region) and K-chains were amplified by the PCR, as described (32). To increase the efficiency of restriction enzyme cutting of PCR-amplified material and subsequent library
construction, a number of extension primers were used, as described (40).
These oligonucleotides contain a poly(GA) tail 5’ to the original primer
sequences, increasing the number of bases between the cutting site and
Fab lithe end of the molecule. The subsequent construction of 1 g G l ~
braries using the pComb3 m13 surface display system has also been
described previously (33,34,36). In brief, the amplified light chain DNA
was cut with the restriction enzymes Sac1 and XbaI and ligated with
SucIiXbuI-linearized pComb3 vector for 3 h. The ligated material was
purified and transformed by electroporation into 200 p,l Escherichia coli
XL1-Blue cells. After transformation, the culture was grown overnight
first in SOC medium and then superbroth (SB) medium containing carbenicillin and tetracycline. For cloning of the heavy chain, phagemid
DNA was digested as described above, except that the restriction enzymes SpeI and XhoI were used. The transformed final IgGlK Fab library
was grown in SOC medium for 1 h at 37°C after addition of SB medium
containing carbenicillin (50 pg/ml) and tetracycline (10 pg/rnl). After
3 h, helper phage VCS-m13 (10” plaque-forming units) was added, and
the culture was shaken for an additional 2 h. Kanamycin (70 pg/ml) was
then added, and the culture was incubated at 30°C overnight. The supernatant was cleared by centrifugation (4000 X g for 20 min) at 4°C. Phage
were precipitated by addition of 5% polyethylene glycol and 0.15 M
NaCl and incubation on ice for 30 min followed by another centrifugation. Phage pellets were resuspended in PBS buffer containing l% BSA
and microcentrifuged for 3 min to pellet debris.
Amplification of Ag binding phage through
library panning
Panning of the combinatorial libraries without epitope masking was conducted as described previously (36). In brief, four microtiter wells were
coated overnight with 0.5 &well recombinant gp120, W strain (gp120
BRU, American BioTechnologies, Cambridge, MA) (41) in 0.1 M bicarbonate buffer, pH 8.6, at 4°C. Plates were washed three times with
water and blocked with PBS containing 3% BSA for 1 h at 37°C. The
BSA was discarded, and SO p1 phage resuspended in PBS were added to
each well and incubated for 2 h. For Ab masking, the wells were previously incubated with the purified Fab fragment L42 and b3 ((37) a Fab
with an aminoacid sequence nearly identical to L28) in a concentration of
40 Fg/ml for 1 h at 37°C. One-half of the volume was then removed
before adding 50 pl of phage. Unbound phage were removed by vigorous
washing 10 times with PBS containing 0.05% Tween 20 (PBS-Tween).
Bound phage, enriched for those bearing Ag-binding surface Fabs, were
eluted with acid. The eluted phage were amplified by infection of E. coli
and superinfection with M13 helper phage. The panning procedure was
repeated 4 times, following which soluble Fab fragments were obtained
by excision of the cpIII gene, achieved by NheI and SpeI digestion and
subsequent ligation of the compatible cohesive ends of the vector.
ELISA analysis of soluble Fab fragments
Fabs were prepared as bacterial supernatants through a freeze-thawing
procedure, as reported earlier (33, 34, 36). To assess specificity, supernatants were screened in an ELISA system (36). Coating of ELISA wells
with gp120 at a concentration of 0.1 Fg/well and subsequent blocking
were conducted as described above. To assess specificity, Fabs were
tested against BSA, OVA, human placental DNA (all from Sigma Chemical Co., St. Louis, MO), and transferrin. Briefly, supernatants were incubated for 2 h at 37°C. Plates were washed 10 times with PBS-Tween,
and bound Fabs were detected with an alkaline phosphatase (AP)-labeled
goat anti-human IgG F(ab’), Ab (Pierce, Rockford, IL) diluted 1 5 0 0 in
PBS, visualized with nitrophenol substrate (NPP substrate) (Sigma
Chemical Co.), and read at 405 nm. Donor sera from the donor was
evaluated for the presence of specific Abs against the gp120 by the Same
ELISA. To investigate whether the epitopes recognized by the Fab fragments were conformational, gp120 wasdenatured and reduced by boiling
for 5 min in PBS containing 1%SDS and 50 mM DTT before 10-fold
dilution into PBS containing 1%Nonidet P-40 to the concentration used
(0.1 pg/well). Native or denatured gp120 was then captured on a solid
phase via the carboxyl terminus by using sheep polyclonal Ab D7324
(Aalto Bioreagents, Dublin, Ireland). A murine Ab BAT-085 (17), which
The Journal of Immunology
has been shown to react almost as well with denatured gp120 as with the
native molecule. was used as a control.
Purification of Fabs
gpl20-binding Fabs werepurified as previously described (38) with minor modifications. In brief, E. coli containing appropriate clones were
inoculated separately into 1-liter cultures of SB containing carbenicillin
(50 pg/ml), tetracycline (10 pg/ml), and MgCl, (20 mM), and grown at
37”C, with shaking,for 6 h. Protein expression was then induced with 2
mM isopropyl P-o-thiogalactopyranoside and grown at 30°C overnight.
Soluble Fab was purified from bacterial supernatants by affinity chromatography, using a sheep anti-human matrk (Schleicher & Schuell, Keene,
NH). The column was washed with a PBS solution and Ab-eluted in 0.2
M glycine-HC1 buffer of pH 2.2 and immediately brought to neutral pH
with 1 M Tris-HC1, pH 9.0.
895
substrate. The CD4-Ab competition experiment was alsoreversed in format: soluble CD4 in 10-fold dilution (lo”, to
M) was incubated
for 1 h at 37°C with immobilized gp120 before Fab at a fixed concentration, which, in earlier titration experiments, gave 75% of maximum
binding, was added and incubated for another 2 h. After washing with
PBS-Tween, bound human Fab were detected with A P anti-human IgG
F(ab‘), and visualized as described above.
Inhibition €LISA to estimate Fab binding affinities
Relative Fab binding affinities were estimated by competition ELISAs, as
described previously (12). gp120was coated onto ELISA wells and
blocked with BSA, as described above. Fab fragments, which had been
determined by titration experiments to give 75% of maximum binding,
were incubated with free gp120 (10”’ to 10” M) for 2 h at 37°C.
Bound human Fab were detected as described above.
Nucleic acid sequencing
Nucleic acid sequencing was conducted on a 373A automated DNA sequencer (ABI, Foster City, Ca) by using a Taq fluorescent dideoxy terminator cycle sequencingkit (ABI). Primers for the elucidation of heavy
chain sequence were SEQGz(S’-GTCGTTGACCAGGCAGCCCAG-3’)
hybridizing to the (+) strand and the T3 primer (S’-A’ITAACCCTCAC
TAAAG-3‘) hybridizing to the (-) strand. For the light chain, SEQKb
primer (5’-ATAGAAGTTG’ITCAGCAGGCA-3’)
and KEF primer (5’GAATI’CTAAACTAGCTAG’ITCG-3’)were used, binding to the (+)
and (-) strands, respectively.
Alkaline-phosphatase labeling of Fabs
The Abs L39, L40, L78, L28,and L42 were labeled with AP by using the
two-step maleimide method (42). In brief, the heterobifunctional crosslinker (sulfosuccinimidyl 4(N-maleimidomethyl) cyclohexane-l-carboxylate) (sulfo-SMCC) in approximately 50-fold molar excess wasallowed
to react with primary amines on calf intestinal AP for 30 min at room
temperature to form a stable amide bond, and the maleimide-activated
AP was subsequently separated by gel filtration. To form sulfhydryl
groups on the Fab fragments, N-succinimidyl-S-acetylthioacetate(SATA) in
approximately 50-fold molar excess was allowed to react for 30 min at room
temperature with 1 ml(200 to 700 pg/ml)of Fab in 100mM phosphate, 100
mM EDTA buffer, pH 7.1, and then transferred to a vial containing 5 mg
hydroxylamine-hydrochloride.After incubation for 2 h at room temperature,
the deacetylated Fab derivative was separated from hydroxylamine-HC1 and
byproducts by gel filtration. Finally, 0.7 ml of SATA-derivatized Fab (approximately 0.4 mg/ml) was incubated with 1mg of malehide-activated AP
for 2 h. Labeled Abs were tested for reactivity against gpl20, BSA, and
OVA to assure that the binding specificity was not affected by the labeling
procedure.
Fab competition ELISA
Cross-competition experiments wereperformed between the directly APlabeled and unlabeled human Fabs for binding to immobilized gp120. In
each case, a fixed concentration of labeled Fab, which, in earlier titration
experiments, had been determined to give 75%of maximum binding, was
incubated with gp120 and unlabeled Fab in twofold dilution steps (0.01
to 100 pg/ml) in PBS for 2 h. The wells were washed, and bound M labeled Fab was detected with NPP substrate.
CD4 competition EL /SA
gp120 was coated onto ELISA wellsand blocked with BSA asdescribed
above. Recombinant Fab in twofold dilution (from 0.3 to 100 pg/ml)
were incubated 1 h with the immobilized gp120 before soluble recombinant CD4 (AIDS Research and Reference Reagent Program, Division
of AIDS, National Institutes of Health, Bethesda, MD) at a fixed concentration of 1 p d m l was added and incubated for an additional 2 h at
37°C. After 10 washes with PBS-Tween, a mouse anti-CD4 mAb Q425
(42) (AIDS Research and Reference Reagent Program, Division of
AIDS, National Institutes of Health) was added and incubated for 1 h.
The wells were again washed with PBS-Tween, incubated with an APlabeled goat anti-mouse IgG F(ab’), (Pierce), and visualized with NPP
Surface plasmon resonance to measure Fab
binding affinities
The kinetics for Fab binding to recombinant gp120 (MN or W strain)
were determined by surface plasmon resonance-based measurements using the BIAcore instrument (Pharmacia, Piscataway, NJ). The sensor
chip was activated for immobilization with N-hydroxysuccinimide and
N-ethyl-N’-(3-diethyl aminopropyl) carbodiimide. gp120 was coupled to
the surface by injection of 50 p l of a SO-pg/ml sample. Excess activated
esters were quenched with 30 pI ethanolamine, 1 M, pH 8.5. Typically,
4000 resonance units were immobilized. Binding of Fab fragments to
immobilized gp120 was studied by injection of Fab in a range of concentrations (0.5 to 80 pglml) at a flow rate of 5 pl/rnin. The association
was monitored as the increase in resonance units per unit time. Dissociation measurements were acquired after the end of the association phase
but with a flow rate of 50 pl/min. The binding surface was regenerated
with HCI, 1 M NaCI, pH 3, and remained active for 20 to 40 measurements. The association and dissociation rate constants, k,, and k,, were
determined from a series of measurements, as described (43-45). Equilibrium association and dissociation constants were deduced from the rate
constants.
Deglycosylation of gp120
The sequential removal of sialic acid and galactose from gpl20 coated
onto microtiter plates was conducted as previously described (46, 47).
Briefly, gp120 microtiter wellswere coated overnight, as described
above. After washing, the wells were treated with 100 mU/ml sialidase
2 h and thereafter rinsed with 50
(Behringwerke) (50 pl/well) at 37°C for
mM sodium acetate buffer, pH 4.5, before exposure to 25 mM sodium
periodate in 50 mM sodium acetate buffer, pH 4.5, for 1 h at room
temperature. Alternatively, the plates were treated with 10 U/well E. coli
/3-galactosidase (Sigma Chemical Co., grade 1% in 0.1 M phosphate buffer/l mM MgCl, for 24 h at 37”C, after sialidase digestion. After washing and
blocking with 3% BSA in PBS for 1h, the plates were incubated with Fabs
to gp120. Bound Fabs were visualized as described above.
Competition with murine V2 and V3 Abs
Cross-competition experiment were performed between the human Fabs
and two murine anti-V2 mAbs, SC258 and 684-238 (17) (kindly provided by Dr. Gerry Robey, Abbott Laboratories, Irving, TX), and with
two murine anti-V3 mAbs, RN3-50.1 and IIIB-V3-13 (48,49) (AIDS
Research and Reference Reagent Program, National Institutes of Health).
Coating of gp120 was conducted as described above. Competing Ab at a
concentration 100 times that giving 75% maximum binding in previous
titration experiments was incubated with the human Fab for 2 h after
washing and detection of the human Fab, as described above. The assay
was also reversed so that the human Fab was added at a concentration 10
times and 100 times that giving 75% maximum binding in previous titration experiments. The murine Ab was detected with an ”labeled
secondary Ab. In each case, controls with no competing Ab and irrelevant Ab were included.
896
Binding of Fabs to gp120 mutants
COS-1 cells were transfected with pSVIIlenw plasmid expressing either
wild-type or mutant HIV-1 (HXBc2) envelope glycoproteins. After 48 h
incubation, the gp120 in the culture supernatant (supplemented with 1 %
Nonidet P-40 detergent) was captured onto a solid phase using a sheep
anti-gpl20 Ab D7324, as described elsewhere (17, 30). A fixed concentration of recombinant Fab, determined by previous titration curves to
give approximately 75% of the level of binding at saturating Fah concentration, was added in TMTSS buffer (17, 30). After incubation and
washing, bound Fabs were visualized by using AP-conjugated anti-Fab
Ab, followed by the AMPAK amplification system (Dako Diagnostics)
(50). Because the level of binding was nonsaturating for the detection
system used (OD,,, values of 0.3 to 0.8 for most Fabs), this method
measures both enhancing and inhibitory amino acid substitutions. As a
reference, the binding of a pool of HIV seropositive serum to each mutant
was determined. The binding of a Fab to each mutant was tested in
triplicate and the mean value was expressed as a binding index, which is
the ratio of OD,, for the Fab to that for the reference serum corrected for
background (mock transfection supernatant). The average value of this
ratio for the whole mutant panel was calculated. Ratios deviating <0.5
times or >1.5 times from the mean ratio were considered significant
inhibitory or enhancing amino acid substitutions, respectively.
HIV-microplaque neutralization assay
The assay to determine neutralizing Ab titer was conducted as described,
with minor modifications (51). In brief, Fabs were 3-fold serially diluted
in 50% assay medium and 50% NHPP and preincubated in quadruplicate
)
20 plaque-forming units (pfu) of
with equal volume (25 ~ 1 containing
HIV-1 (MN or LA1 strain) per well for 18 h in 96-we11 microtiter plates
in 5% C02/95% air atmosphere at 37°C. Thereafter, 90,000 MT-2 cells
in a volume of 25 @I were added to each well and incubated an additional
h at 37°C. Assay medium (75 @I) containing 1.6% SeaPlaque Agarose
(FMC Bioproducts, Rockland, Maine) heated to 39.5"C was added to
each well and the plates were immediately centrifuged (500 X 8 ) for 20
min at 20°C to form cell monolayers. Plates were incubated for 6 days at
37°C and then stained with 50 &ml propidium iodide in PBS. After 24
to 48 h, fluorescent plaques were counted on a transilluminator (304 nm).
Each Fab was run at least twice against each strain, and the most potent
neutralizers were titered. The neutralizing titer was defined as the concentration of Fab required to give a 50% reduction in plaque numbers as
compared with controls containing no Fab. This dilution was interpolated
between data points, Within each run, the intrinsic statistical error of the
interpolated titers averages 2 30%. A well-characterized neutralizing
Fab was included in each test as an internal control.
Neutralization o f HIV- 1 by using an envelope
complementation assay
The ability of recombinant Fabs to neutralize the MN isolate and the
HXBc2 molecular clone of the HTLV-IIIB (LAI) isolate were assessed in
an envelope complementation assay (52). Briefly, COS-1 cells were cotransfected with a plasmid expressing envelope glycoproteins and a plasmid containing an env-defective HIV-1 virus encoding the bacterial
chloramphenicol acetyltransferase gene. Equal fractions of the cell supernatants containing recombinant virions were incubated at 37°C for 1
h with varying concentrations of Fab before incubation with Jurkat cells.
Three days post-infection, Jurkat cells were lysed, and chloramphenicol
acetyltransferase activity was measured.
Results
Library construction and Ab selection
An IgGlK Fab library was prepared from the bone marrow
of a long-term asymptomatic HIV-1seropositive male donor. The library, designated L, consisted of 2 X lo6 members. Donor serum, taken concomitantly with bone marrow samples, showed serologicactivity with an Ab titer of
1:400 for gp120 LAI. The library was panned against recombinant gp120 LA1 coated onto microtiter wells to en-
NEUTRALIZING HUMAN HIV-1 AbsFROM PHAGE LIBRARIES
rich for specific Ag-binding clones, Four rounds of panning resulted in a 50-foldamplification of elutedphage.
Phagemid DNA was prepared from the second, third, and
fourth rounds of panning and the gene 111 fragment was removed by treatment with the enzymes NheI and SpeI followed by religation. The reconstructed phagemid was used to
transform XL1-Blue cells to produce clones secreting soluble
Fab fragments.
From the fourth round of panning, 15 randomly picked
clones were grown in 10 ml culture, and the supernatants
containing the Fab fragments were screened for reactivity
with recombinant gp120 LA1 in an ELISAsystem. All
clones examined reacted with gp120, but not with BSA.
From the second and third round of panning, 7 of 15 and
12 of 15 clones showedreactivity against gp120. Sequence
analysis of the variable regions of the heavy chain revealed that 10 of the 34 positive clones were unique. As
shown in Figure 1A, the sequences could be organized into
four distinct heavy chain types. The sequence discrepancies within the groups beginning with L28 and L4l are
very small and could arise from the PCR or the reverse
transcription method. However, there is a marked difference of 8 amino acids between the two clones in the group
beginning with L42that is more likely attributed to in vivo
somatic mutation. The variable light chains (V,) of the
gp120 binding clones were also sequenced and organized
into the groups defined by Figure lA, as shown in Figure
1C. As also observed with the gpl20-binding Fabs from
the M library (37), extensive chain promiscuity was seen;
different V, were able to combine with the same, or a very
similar, V, without affecting the Ag-binding ability in the
gp120 ELISA, as exemplified in the group beginning with
L28. We also observed the opposite result on one occasion: different V, binding (L28A, LA2) to the same V,. In
the further characterization of the Ahs, we chose one as
representative from each group, namely L28, L33, L42,
and L52. L28 and L42 dominated the Ab repertoire obtained, constituting 21 of 34 gpl20-binding clones.
Library panning after epitope masking by Fabs
To examine the possibility of obtaining a larger panel of
Abs reacting with different regions of gp120, Fabs corresponding to the two dominant clones from the initial panning of the L library were purified and used to mask their
respective epitopes on gp120, before repeating panning of
the L library. After four roundsof panning, 15 of 20 clones
were positive. Sequence analysis of the heavy chains revealed that all the clones obtained contained V, sequences
differentfrom those obtained without epitope masking
(Fig. 1B). These new clones could be organized into 4 new
groups of Abs according to their V, sequence. The V,
sequences of the clones are shown in Fig. 1D. For further
characterization of these Abs, we chose one representative
from each group (L39, L40,LA1, and L78).
897
The Journal of Immunology
CDRl
FR4
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LESGPGLVKPSQTLSLTCWSGASVSS
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RVTMSVDRSKNQFSLKLSSVTMDTAVYYCAR
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~ 4 1
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...
HVWDCGSYQNYRDS'YKG
RFT1SRDNSKNTLYLQMNSLRAEDTA~'YYCAP. U T F G V L R L L K G W F D P
L78
LEESGGGVVQPGRSLRLSCAVSGFIFD
CLONE
GYYWS
WIRQHPGLEWIG
HZQGTL'r'T:SS
.............................................................................
. . . . . . . . . . . . . . . . .I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.......................................... T
......R . . . .
..
FR2
NSGMN
WRQAPGKGLEWJA
VISYDAQHQDYGDSVRG
CDR2
CDRl
FR1
RFTISRDNSKNTLFLQMNRLRTDDTA'JYFCAK
FR 3
FR4
GAWGLGYSYMDV
NGSGTAVWSS
CDR3
C
DASTRAT
G......
L G . N . .S
G.. . . .
GIPDRFSGSGSGTDFTLTISRVEPEDFAVYYC
. . A . . . . . . . . .E . . . . . . S L Q S . . . . . . . .
.'I. . . . . . . . .Y . . .K . . . . . A . .VGT . .
. . A . . . . . . . .E . . . . . . S L Q S . . . . . . .
QQYGSFPIT
. . .N N R . Y .
M.hLQT.Y.
. . .NNR . Y .
RASQSVSSNLA
MAELTQSPSPPCLCLQGKEPTLSC WYQQKFGQAPRLLIY
DTSNRAT
GIPARFSGSGSGTDFTLTISSLEPEDSAVYYC
QQYGSRPGYT
FGQGTKLEIKRTJA
WYLQKFGLSPQLLW
MAEL'IQSPLSLPVTPWRAGVSISC WSSQSLLHSNGYNYLD
. . . . . A . . . . .H . . . . R P . . . Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LGSNRAS
MQhLQTPYT
FGQGTRLEIKRTIA
V......
GVPDRFSGDTSGTYFTLKISRVEAEDVGVYYC
.S . . . . . .SG ......................
QASQDI'JNRLN
~ E L T Q S P S L P V C A S V G D R V T IWYQLKPGRAPYLLIY
~
DTSNLEE
G'IPARFSGDTSGTEFTLTISSLQPEDVATYYC
Z Q T S AFFGPQLGTT R L E I K R T I A
WYQHKPGMPNLLIY
TASRLRS
i~lPSRFSGSGSGTDFTLTIISLQPEDFATYYC
LQSYGPPPT
FGQGTKVFI'KRTIA
GVPSRFSGDTSGTEFTLTISSLQPEDVATYYC
Q Q T S AFFGPQLGTT R L E I K R T J A
G'JPSRFSGSGSGTDFTLTISSLQPEDFATYYC
QQGYSTPEYT
FGQGTKLEIKRWA
G'IPARFSGSGSGTDFTLTISSLQPEDFATYFC
QQFKIYPLT
FGGGTKVEIRRTJA
L28
L28A
L286
L28C
MAELTQSPGTLSLSPGARATLSC
. . . . . . . . D . . . . . RGE . . . . . .
. . . . . . LS.PVT..EP.SI..
. . . . . . . .D . . . . .R . K . . . . . .
L33
L42
L42A
L 52
WYQQKPGQTPRLLIY
RASQSVSSNLA
...........
. . . . . . . .A , . . . . .
. . . LLHSNGYNYLD . .L . . . . L S . Q . . V .
...........
. . . . . . . .A , . . . .
WS
FGQGTRLDLKRWA
.... W E 1 . . . . .
. . . . .K . E I . . . .
. . . . .WJEI . . . . .
.....................
~
D
L39
MAEL~SPPSLSr?F'jGDRVTIAC
RASQPISNSLN
L40
MAELTQSPSSLSASVGDRVTISC
PASQNIASRLA
WYQQKPGMPNLLIY
DASNLW
L41
L41A
L4l6
MAELTPSPSSLSASVGDRVTFAC
. . .V . . . . . . . . . . . . . . . . I . .
. . . . . . . . T . . . . . . . . . . .I . .
RASQSISNYLN
WYQQKVGWFKLLIY
AASTLQS
L78
MAELTQSPSSLSASVGDRLTITC
RASQDISEYLA
WYQQKPGFAPKLLIY
AASTLQS
...........
...........
............................................................................
............................................................................
FIGURE 1. Amino acid sequences of variable heavy ( A and €3) and light ( C and D ) domains of anti-gpl20 Fabs retrieved
without ( A and C ) and with (BandD ) epitope masking. The N-terminal sequence LEQSG arises from the VH1 a and LEESG from
the VH3a primers (36). Identity with the first sequence in a group is indicated by dots.
To confirm that the new Abs selected were not related to
some irreproducibility in the method, the L library was
panned again against unmasked gp120 LAI. After four
rounds of panning all of the 9 positive gpl20-binding
clones had sequences identical with those obtained in the
first panning experiment without epitope masking. The L
library was also panned against gp120 of the SF2 strain.
After four rounds of panning, five positive clones were
sequenced. Two clones had V, sequences similar to L28,
two had sequences similar to LA2 and the last sequence
was identical with L33: all of these clones were obtained
before epitope masking using gp120 of the LA1 strain. The
results underline the reproducibility of the method and
also confirm that the primarily selected Abs are strain
cross-reactive.
data show that binding to gp120 of Fabs L39, L40, or L78
was not inhibited by pooled L28 and L42. Binding of L78,
and especially of L40,was in fact enhanced by previous
binding of L28 and LA2. In contrast, the binding of Fab
L41 was inhibited by L28 and L42 (Table I).
The two Abs used for masking were also AP-labeled to
insure that the previous competition results were not a result of labeling artifacts. Competition ELISA with the labeled masking Ab and Fabs L39, L40, L41, and L78 are
depicted in Figure 2B. In this assay, LA1 blocked the binding of L42. In contrast, Fabs L39, L40, and L78 did not
inhibit L42. Similar results were obtained with L28 (data
not shown). This implies that L39, L40, and L78 are directed to a different site ongp120 than L28 and L42,
whereas L41 is directed to a closely related epitope.
Ab competition studies
Ab characterization and epitope mapping
To determine if the Abs obtained after epitope masking
were directed against novel epitopes on gp120, the Fabs
L39, LAO, L41, and L78 were labeled with AP by using the
two-step maleimide method. Competition ELISA with labeled Ab and masking Abs is depicted in Figure 2 A . The
To estimate the affinities of the Fab fragments for gp120
LAI, inhibition ELISAs were conducted by utilizing soluble recombinant gp120 to compete with microtiter wellbound gp120 for Fab binding. As shown in Figure 3, all
the Abs had apparent affinities of 1 X lo7 to 5 X lo8 M-'.
NEUTRALIZING HUMAN HIV-1 Abs FROM PHAGE LIBRARIES
A
0
0
!.a&€&
CI
0
II
0
,001
L39
L40
L41
L78
E
II
II
IIIInIIm
.01
.1
1 1 0 01 0
1000
pg/ml competing Fabs (L28 and L42)
B
CI
0
II
Conmetitor
CI
II
0
.001
L39
L40
L41
L78
.01
.1
1
10
100
1000
pg/ml Competing Fab
FIGURE 2. A ) competition of AP-labeled L39, L40,L41, or
L78 with unlabeled L28 and L42. B ) competition of AP-labeled L42 with unlabeled L39, L40,L41, or L78. In each
case, a fixed concentration of the labeled Fab wasused,
which had been determined by titration experiments to give
75% of maximum binding. Bound labeled Fab was visualized with nitrophenol substrate.
For more detailed binding analysis, several Fabs werechosen for further study using real-time biospecific interaction
analysis (BIAcore). Association constants (K,)and dissociation constants ( K d ) for Fab binding to gp120 MN and
LAI strains are presented in Table 11. Binding constants for
LAI correlated to those obtained by inhibition ELISA,
with K, in the range of 5 X lo7 to 5 X 10‘ M-‘; i.e., Kd
in the range of 2 X lo-’ to 1 X
M. Fabs L41 and
L42, both directed to the CD4bs, showed equivalent binding constants for MN and LAI, whereas Fab L28 showed
tighter binding to LAI. All previously tested CD4bs Fabs
have also shown tighter binding to LAI (data not shown).
Of the Abs obtained with epitope-masking,
LA1 had the tightest binding of all the Fabs studied with a Kd for gp120 in the
nanomolar range. Fabs L40 and L78 had slightly lower affinities and bound gp120 LAI more strongly than MN.
To investigate the ability of the panel of Fabs to compete with soluble CD4 for gp120 binding, competition
ELISAs with gp120 coated onto microtiter wells were
conducted. Fab in increasing concentration was allowed to
react with gp120 before a constant amount of soluble CD4
was added. All Fabs retrieved without epitope masking,
and Fab L41 retrieved with epitope masking, inhibited
binding of CD4 to gp120. In contrast, L39, LAO, and L78
did not inhibit CD4 binding. The competition experiment
was alsoreversed by incubating increasing amounts of soluble CD4 with Fabs at a fixed concentration giving 75% of
maximum binding. In this assay, all the Fabs in the panel
were competed by soluble CD4 (Fig. 4). The most favored
interpretation of these results is that Fabs L28, L33, L41,
L42, and L52 are directed against the CD4bs, whereas
Fabs L39, LAO, and L78 are directed against an epitope
distinct from the CD4bs. However, the binding of soluble
CD4 to gp120 induces a conformational change of gp120
which abrogates the binding of these Abs. The same conformational change in gp120 does not seem to be induced
by the anti-CD4bs Abs described above. Instead the binding of anti-CD4bs Abs actually enhances binding of LAO
and L78 to their epitopes (Table I).
The panel of Fabs was further assessed for reaction with
gp120 LAI, either in native form or after denaturation by
boiling in the presence of DTT and SDS. As shown in
Figure 5 and Table I, the binding of all of the Fabs was
reduced by denaturation of the gp120 LA1 molecule. However, whereas denaturation totally abolished reactivity
with the CD4bs Fabs, Fabs L39, L40, and L78 still exhibited significant binding.
To further characterize the epitopes recognized, recombinant gp120 LAI was treated with neuraminidase/&galactosidase or neuraminidase/periodate to remove (pl-4)linked galactose from N-linked sugars. As shown in Figure
6 and Table I, binding of all the Fabs obtained before Ab
masking and L41 obtained after masking, was absolutely
dependent on this glycosylation. In contrast, Fab L40
binding was only partially affected by removal of peripheral monosaccharides, and binding of Fabs L39 or L78
was unaffected by either sialidaselp-galactosidase or periodate treatment.
A panel of gp120 mutants expressed in COS-1 cellswas
also used to characterize the epitopes recognized by the
panel of combinatorial human Fabs. This panel of mutants,
generated by mutations in the env gene of the HXBc2
HIV-1 molecular clone and cloned into the pSVIIIenv
plasmid, have previously been used to characterize the
CD4bs and panels of Abs to gp120. The OD value of Fab
binding to a given mutant was compared with the binding
The Journal of Immunology
899
Binding pattern of the panel of human recombinant Fabs
Table I.
Effect of CD4bs
Ahs o n Fab Binding
to gp120
Fab Designation
Retrieved without masking
L28
L33
L42
L52
Retrieved wittmasking
inhibit
L40
L41
L78
120
inhibit
inhibit
inhibit
inhibit
inhibit
inhibit
inhibit
inhibit
L39
n o effect
enhance
inhibit
enhance
inhibit
no
Effect of Fah o n
sCD4 Bindlng
t o gpl20
no
no
no
no
no effect
weak
inhibit
inhibit
weak
effect
inhibit
I
-
DL28
DL33
DL42
7 DL52
"
t DL39
DL40
DL41
1DL78
I
-12
-11
-10
-9
-8
inhibit
inhibit
inhibit
inhibit
noweak
effect
,
-13
Effect of sCD4 on
Binding
Binding
to
to
Fah Binding
Denatured
Deglycosylated
gP120
to gpl20
-7
-6
Log [competing gp120 IllB]
FIGURE 3. Relativeaffinities of recombinant human Fabs
for gpl20 LA1 as measured by inhibition ELISA.
of the Fab to the wild-type gp120 and expressed as a binding ratio. Fabs L28 and L42, were first studied (Table 111)
and their binding to HXBc2 gp120 in the ELISA system
was abolished or strongly impaired by amino acid substitutions previously shown to abrogate or greatly reduce
CD4 and CD4bs Ab binding (10, 53), i.e., 368 D/R, 368
Dm, 370 E/R, and 257 T/R. In addition, L42 binding was
moderately reduced by changes 266A/E and 477 D/V, and
L28 binding was also reduced by changes 370 EIQ, 475
M/S, 102 E L , and 463 NID. Binding of L28 wasenhanced
by changes at 45 W/S, 298 R/G, 381 E/P, 382 FIL, 420
I/R, 435 Y/H, and 435 Y/S and the removal of the whole
V3 region, whereas LA2 binding was significantly enhanced by changes at 381 E/P and 382 FIL.
The Fabs obtained after the epitope masking were then
studied. Substitutions at 368 D/R, 370 EIR, and 257 T/R
abolished or markedly reduced Fab L78 binding. However
in addition, changes within the V2 loop also strongly impaired binding of this Fab, i.e., 1521153 GE/SM, 1831184
PI/SG, 191/193, and YL/GS, and the excision of the whole
gP120
-
Sensitivity to v 2
Mutations
-
no
no
no
no
+
++
Yes
Ye5
no
Yes
-
Vl/V2 loop structure also moderately impaired the binding. A substitution at 262N/T in the C3 region abolished
the binding of L78 and a substitution at 314 G/W at the tip
of the V3 loop moderately inhibited L78 binding. In contrast, substitutions in the C4 and C5 region (420 I/R, 435
YIS, 435 Y/H, 438 P/R, 475 MIS, and 495 G/K) enhanced
L78 binding. As shown in Table 111, Fabs L39 and L40
showed similar binding patterns to L78, with only minor
differences. Fab L41, retrieved after epitope masking but
competitive with masking Abs, was also tested against the
gp120 mutants. Although this Fab showed many similarities with L28 and L42, such as an inability to bind to
mutants with substitutions at 257 T/R, 368 D/R, 368 D E ,
370 E/Q, and 370 E/R, L41 binding was also completely
abolished or significantly reduced by substitutions at 113
D/R, 256 S j Y , 384 Y E , and 421 WL. These substitutions
either enhanced or had no effect on the binding of L28 and
L42. Substitutions at 380 GJF and 381 EIP and the removal
of the whole V3 region enhanced LA1 binding.
Additional evidence that Fabs L39, L40, and L78 are
influenced by the V2 region in binding to gp120 was provided by competition assays between these Fabs and two
previously described murine anti-V2 mAbs (Fig. 7). As
shown in Figure 7a, Abs SC258 and 684-238 both inhibited binding of Fabs L39, L40, and L78 to gp120; in the
reverse format, these Fabs inhibited SC258 and 684-238
binding to gp120 (Fig. 7, b and c). In contrast, SC258 and
684-238 Abs did not inhibit LA1 binding to gp120, nor did
Fab L41 block binding of these anti-V2 Abs. The panel of
human Fabs were also tested against two murine anti-V3
loop mAbs, RN3-50.1 and IIIB-V3-13, and no competition was observed (data not shown).
We have previously reported on a very potent neutralizing Fab, b12, which, in mutant studies, has been mapped
to the CD4bs (11, 54). However, b12 binding has also
been found to be reduced by substitution at 1831184 PI/SG
(binding ratio = 0.33) and excision of the VlfV2 loop
(0.23) (54). Because of this V2 dependence, we investigated the similarities and differences between L78 and
b12. In competition experiments, b12 did not compete
NEUTRALIZING H U M A N HIV-1 Abs FROM PHAGE LIBRARIES
900
Table II.
Kinetic constants and affinity constants for the binding of selected Fabs to LA/ and MN gpl20 measured by surface plasmon
resonance'
Fab
gpl20 LA1
L28
L42
L41
L40
L78
gpl20 MN
L28
L42
L41
L40
L78
a
K, ("'1
(M)
Kd ? SD (nM)
Kon
KO,
1.98 X 104
2.50 x 104
7.4 x 104
1.80 X 104
1.90 x 104
5.80 x 10-5
1.40 X 10-4
1.30 x 10-4
1.14 X 10-4
I 20 x 10-4
3.40 X lo8
1.80 X 10'
5.7 x l o 8
1.60 X lo8
1.10 x lo8
2.90 X 10-9
5.6 x 10-9
1.70 X 10-9
6.10 x 10-9
9.50 X
2.9 (0.33)
5.6 (0.06)
1.7 (0.1 3)
6.1 (0.56)
9.5 (0.05)
x
x
4.90 x
7.4 x
7.9 x
1.30 x 1o - ~
9.5 x 10-5
1.15 x
8.80x 1 0 - ~
1.77 x
6.00 x 10'
1.70 X l o 8
4.30 X 10'
8.40 x l o 7
4.50 x l o 7
1.70 X
6.00 x
2.40 x
1.20 x
2.20 x
17 (0.5)
6 (0.5)
2.4 10.2)
12 (0.14)
22 (2.2)
7.6
1.60
103
104
104
103
103
Kd
lo-@
10-9
10-9
10-8
10-8
The equilibrium association and dissociation constants were calculated from the experimentally determined kinetic constants whereK, = k,Jk,, and
with L78 forbinding to gp120(Fig. 7d), nor did b12 comPete with the two murine anti-V2 region Abs (SC258 and
684-238). A whole IgG construct of b12 was used for
these experiments to allow detection of b12 by a labeled
anti-human IgG Fc Ab. In CD4 competition experiments,
preincubation of b12, in contrast with L78, inhibited CD4
binding to gp120. Finally, b12 binding, in contrast with
L78, was abolished after gp120 denaturation as well as
sialidaselp-galactosidase and sialidase/periodate deglycosylation of gp120 (data not shown).
Virus neutralization
Affinity-purified Fab from each of the eight Abs in the
panel were examined for neutralizing ability in infectivity
assays by using MN and LAI strains of HIV-1. Neutralization was determined as the ability of the Fab fragments
to inhibit infection, as measured by aplaque reduction
assay using MT-2 cells. As shown in Table IV, a significant difference in the neutralization ability of the different
anti-HIV-1Fabswerefound.Three
of the CD4bs Abs
(L42, L41, and L28) showed 50% inhibition of the LAI
strain of HIV-1 at concentrations below 2 pg/ml and 50%
inhibition of the MN strain below 10 pg/ml. The CD4bs
Fab LA2 was an especially efficient neutralizer, with 50%
inhibition at 0.7 pg/ml and 0.9 pg/ml for the MN and LAI
strain of HIV-1, respectively. On the basis of this limited
initial study, only two of the CD4bs Fabs (L33 and L4l)
seemed to be relatively strain-specific. Of the V2-dependent Fabs, only L78 showed good neutralization ability,
with 50% inhibition at 2 pg/ml and 1.6 pg/ml for the MN
and LA1 strains of HIV-1, respectively.
To further examine the neutralization ability of the Fabs,
those showing potent neutralization in the plaque reduction
assay were analyzed in an envelope complementation assay
using the LAI and MN strain. The CD4bs Ab LA2 showed
50% neutralization at less than 1 pg/ml for the MN and at 1
p d m l for the LAI strain. LA1 showed 50% neutralization at
1 pg/ml for both MN and LAI strains. Finally, the V2-dependent Fab L78 showed 50% neutralization at 10 p d m l for
=
kJk,,.
the MN strain, but did not reach 50% neutralization of the
LAI strain at this concentration.
Discussion
The use of combinatorial libraries displayed on the surface
of filamentous bacteriophage offers an efficient route to
obtain a diverse set of human mAbs from an immune donor (55). However, certain epitope specificities may dominate the cloned Abs because, for example, the epitope is
immunodominant or because Abs to that epitope have a
selective advantage in the panning process arising from a
higher affinity.In this report, we describe an epitopemasking strategy by whichAbs to an extendedset of
epitopes can be retrieved. The strategy has been used to
successfully clone a neutralizing human Ab directed to a
previously undefined conformational epitope influenced
by both the V2 region and the CD4bs of the gp120 molecule on HIV-1 (Table I).
When using recombinant gp120 LAI as the Ag in affinity selection our experience with several libraries constructed from asymptomatic long term HIV-1 seropositive
donors is that the great majority of the retrieved Fabs are
directed against the CD4bs (11,36,37). Two factors probably contribute to this observation. First, the immune response in these patients is strongly directed to this epitope
(56), and second, the retrieved Abs are not selected by
gp120 from the infecting strain, but rather by that of the
divergent strain LAI. An important part of our overall
strategy to retrieve broadly reactive Abs potentially useful
for immunotherapy involves using a strain far from that
generally causing infection in North America, effectively
eliminating strain-specific Abs. This tends to focus Ab selection onto the CD4bs, but even in hypervariable regions
such as the V2 and V3 loops some conserved features are
found (57-60), and the challenge is to retrieve these minor
strain cross-reactive Ab specificities from libraries. For the
V3 loop, we have used V3 peptides (37). A n alternative is
to block the major CD4bs specificity with existing Abs
before affinity selection from the library. This approach
901
The journal of Immunology
I
E
2
VI
3
v)
*
n
0
P
0
1-
-
0
.
"
c
-t
-P-
1
n
"
.
.
-
.
.
L28 L33 L42 L52 L39 L40 L41 L78 BAT-085
L28
L42
L30
L40
L41
L78
FIGURE 5. Binding of recombinantFab fragments to native
(a)and denatured gp120 LAI. The Fabs were used at a
concentration giving 75% maximum binding to native gpl20
in titration experiments.
m)
0
I
I
.1
1
10
100
pglml competing Fab
I
120
IS
v)
0
d
n
1
0
'1
1
I
0,
"0-
-12
-11
0
L28 ~ 3 3~ 4 2 ~ 5~ 23 9L ~ OL ~ I ~ 7 a
L28
L33
L42
I
L41
L70
I
I
I
I
I
-10
-9
- 8
-7
- 6
I
FIGURE 6. Bindingofrecombinant Fabsaftersequential
removal of carbohydrates fromgpl20 LAI. Effect of combined
sialidaselp-galactosidase (H)
or sialidasdperiodate(0)treatment on the reactivityof the panel of Fab fragments as compared with untreated gpl20 4).
-5
log [competing CD4]
FIGURE 4. Competition betweenFabsandsCD4 for binding to recombinant gp120. A) in this set of competition experiments, Fabs in increasing concentration were allowedto
react with gp120beforeaconstantamount of soluble CD4
was added. The binding of CD4 was detected with a monoclonal anti-CD4 Ab. B) in a secondset of competition experiments, soluble CD4 in increasing concentration was allowed to react with gpl20 before a constant amountof Fab
fragment was added. The
binding of human Fab was detected
with a labeled anti-human IgG F(ab'), Ab.
-
has yielded high-affinity strain cross-reactive Abs that recognize a complex epitope dependent on the V2 loop.
Several groups have recently described V2-dependent
regions of gp120 containing neutralizing linear and conformational epitopes using murine mAbs (14-18). It has
been predicted that V2 Abs are also part of the humanAb
response. However, it seems unlikely that the fine speci-
ficity of the Ab repertoire in rodents immunized with recombinant HIV-1 proteins or peptides will be identical
with that in humans infected with the
HIV-1 virus. The
Prevalence of Vz-dePendent Abs inthehumanimmune
response against HIV-1 is difficult to determine merely by
serology. Previous evaluations have used peptides, but, as
indicated previously (17, 61) and by our experience, artifactual binding can occur and informationis limited to the
fraction of the response not dependent on conformation.
Anotherapproach(17) has been to evaluate the ability
of sera from HIV-infected individuals to inhibit binding of
murineV2-dependentmAbs.Serum
from a subset of
HIV-1 seropositive individuals inhibited these murine Abs
(17), but one problem with this approach is that HIV-1
sera contains Abs that enhance as well as those that inhibit
gp120 binding of V2Abs (17). An additional complication
of serologic analysis of HIV-1 infection is that infected
individuals may have high levels of Abs that recognize,
with moderateaffinity,a wide variety of Ags (62). To
902
NEUTRALIZING H U M A N HIV-1 Abs FROM PHAGE LIBRARIES
Relative binding of human
recombinant Fabs to
selected HXBc2 gp120 mutant9
Table Ill.
L78
L42
L41
L40
L39
L28
sCD4 binding to gp120, it might be expected that CD4
binding would not inhibit Fab binding. However, in a second set of competition experiments wherein sCD4was
meincubated with ~ 0 1 2 0 .inhibition of the V2-deoendent
Fabs was observed. This surprisingbehavior has also been
described independently from two studies on murine V2dependent Abs (16, 17) and implies some elements of irreversibility in the interaction of CD4 and gp120. It also
implies that CD4 binding decreases the availability of the
V2-dependent epitope, adding to the body of evidence suggesting CD4-induced conformational changesin a 1 2 0 (63).
Competition experiments between the CD4bs Abs and
the V2-dependent Abs indicated that preincubation of two
CD4bs Abs, L28 and L42, enhanced binding of V2 Abs
LAO, and L78, whereas binding of L41 (CD4bs Ab) was
inhibited. Again, similar results were obtained in the murine system, in that binding of VZdependent murine Abs
to gp120 was enhanced by CD4bs Abs (17), suggesting
that CD4bs Abs induce conformational changes in gp120
thatincreasethe exposure of theV2epitope.Indeed,this
enhanced exposuremay be a key factor in the retrievalof the
V2-dependent Abs after CD4bs masking.It should be noted,
however, that other anti-CD4bsAbs have been reported that
do not enhance binding of V2-dependent Abs (17).
Using a panel of gp120 mutants expressed in COS-1
cells, the binding specificities of the three novel Fabs were
mapped. All were sensitive to V2 region changes and, to a
lesser extent, to excision of the V1W2 loop. These
changes are not believed to produce global changes in
gp120 conformation as they do not generally perturb Ab
binding to the CD4bs (10,30,54) although they do perturb
CD4 binding (21). Perhaps unexpectedly, binding of the
novel Fabs was also sensitive to changes at residues 368
D/R, 368 DIT, and 370 E/R, generally considered essential
for binding of CD4 and CD4bs Abs (10, 53). In addition,
a substitution at 262 NIT in the C3 region abolished L78
binding. None of the previously described murine V2-dependent Abs were sensitive to these latter changes (17).
Binding of the three V2-dependent human Fabs was enhanced by substitutions within the C5 region, as previously reported for V2-dependent murine Abs (17). Further
evidence that the three novel Fabs were dependent on the
V2 region for gp120 binding was provided by competition
between the Fab fragments and the previously described
murine VZdependent Abs, SC258 and 684-238 (17).
From the above, it is clear that the epitope we have
described is distinct from that recognized by the V2-dependent murine Abs characterized to date. In this regard, it
should again be emphasized that the murine Abs have
been generated by immunization with recombinant proteins or peptides, whereas the human Abs arise from natural infection.
Surprisingly, L41, the last of the four Abs retrieved after
panning on masked gp120, competed with the two CD4bs
masking Abs and, in mutant studies, LA1 was mapped to
the CD4bs. The differences between L41, L28,and L42
-1
45 WIS
102 EjL
106 VA
113 D/R
117 KhV
152/153
G E/S M
168 K/L
183/184
PVSC
1921194
YSL/GSS
AV1N2
207 K h V
256 S r /
257 T/R
262 N/T
266 NE
281 A/V
298 WC
314 G/W
A V3
368 D/R
368 D/r
370 EIR
370 E/Q
380 C/F
381 E/P
382 F/L
384 YIE
420 IIR
421 K/L
432 KIA
435 YIH
435 YIS
438 P/R
435 Y/S
438 PIR
457 D/A
463 NID
475 MIS
477 DN
491 IIF
1.60
0.46
0.91
1.40
1.01
0.70
0.35
0.65
0.41
0.53
0.71
0.18
0.65
0.42
0.23
1.13
1.32
0.48
1.03
0.66
1.11
0.30
1.20
0.80
1.03
0.88
1.05
1.28
1.24
0.93
0.68
0.59
0.59
0.83
1.24
0.46
0.78
0.82
0.47
0.18
0.19
0.88
0.13
0.53
1 .OO
0.63
0.83
0.19
0.82
0.29
0.32
0.94
0.93
0.29
1.07
1.15
0.64
0.04
1.06
1.03
0.69
1.60
0.59
1.88
0.18
0.13
0.00
0.18
1.54
2.18
1.72
1.21
1.70
0.82
1.59
1.73
1.93
1.20
1.93
1.20
0.62
0.48
0.13
0.50
0.68
0.53
2.47
0.47
0.41
0.47
0.88
0.18
0.76
0.24
0.88
0.24
0.06
0.00
0.47
1.06
1.29
ND
0.47
1.47
0.76
1.76
2.06
2.59
1.47
2.59
1.47
1.06
1.88
3.41
1.47
1.94
0.52
0.94
0.42
0.06
0.00
0.58
0.65
1.39
0.52
1.26
0.00
0.00
0.00
0.10
1.29
1.23
ND
0.45
1.81
1.19
2.00
2.32
2.58
2.22
2.58
2.22
0.58
1.42
2.06
0.23
1.52
1.16
1.14
0.00
0.02
0.34
0.97
0.75
1.27
0.47
1.64
0.00
0.00
0.00
0.00
1.52
1.44
1.08
0.00
1.28
0.00
1.02
1.17
1.39
1.11
1.39
1.1 1
0.78
0.58
0.89
0.39
1.05
0.95
1 .I4
0.29
0.01
0.72
0.55
0.64
1.04
0.62
1.45
0.01
0.05
0.00
0.97
1.39
1.67
1.55
1.40
1.38
0.83
1.16
1.21
1.18
1.09
1.18
1.09
0.84
0.63
1.03
0.46
0.89
0.54
1.37
0.66
0.15
0.00
0.76
0.93
1.49
0.24
1.17
0.10
0.05
0.00
0.29
0.93
1.22
ND
0.59
1.73
1.37
1.41
1.73
1.83
1.56
1.83
1.56
0.68
1.22
1.54
0.46
1.46
" The following mutants were also included in the test panel but did not
produce a binding index 1 0 . 5 or >1.5 for any of the Fabs tested: 36 VIL, 4 0
YID, 69 WIL, 76 P/v, 80 NIR, 88 NIP, 103 Q/F, 1 13 DIA, 120I121 VWLE, 125
LC, 1761177 FY/AT, 179/180 LD/DL, 252 W, 257 TIA, 267 VL, 269 E/L, MT
33+1, 313 PIS, 356 N/i, 386 N/Q, 392 N/E 397 N/E, 395 WIS, 406 N/G, 420
I/R, 427 WIS, 429 KJL, 430 VIS, 433 AJL, 450 TIN, 456 WK, 457 DIR, 470 PIG,
470 P/L, 485 K
V
j , and 493 PIK. ND, not done.
study the humoral response to such epitopes requires access to human mAbs. Compared with murine V2-dependent Abs, the human VZsensitive Fab fragments showed
many similar characteristics, but also some important differences, as outlined below.
The epitope(s) recognized by the novel Fabs were
probed by a series of competition experiments between
Fabs and CD4 and CD4bs Abs. Competition experiments
between Fabs and sCD4 showed that, whereas all the antiCD4bs Fabs inhibited sCD4 from binding, Fabs L39, L40,
and L78 had no effect, indicating a binding site distinct
from the CD4bs. As Fab L39, LAO, and L78 did not inhibit
1
-
~
~
903
The Journal of Immunology
1.2 -I
1.0
1.6
E
e
1.2
0.6
*
v)
0
*
i
0.8
8
n
0
0.4
0.4
0.2
0.0
0.0
S C 2 5 8 LL3L4L970481
alone + competing recombinant Fabs
L 4 1L 7 8L 4 0L 3 9
1.6
C
1.6
1.2
z
v)
E
0.8
0
0
n
n
*
v
0.4
1.2
v)
0.8
0
0.4
0.0
0.0
IgGlbl2
6 8 4 - 2 3 8 LLLL34749081
alone + competingrecombinant Fabs
alone
SbC
1258
L78
684-2 8
+ competing antibodies
FIGURE 7. a) Binding of Fabs L39, L40, L78 and L41 (in a concentration giving 75% maximum binding) to g p l 2 0 LA1 either
at a concentration 100 times that giving 75%
alone) . ( or competed with mouse anti-V2 Ab SC258 (El) or 684-238 (0)
maximum binding in previous titration experiments. b) Binding of mouse anti-V2 Ab SC258 (at a concentration giving 75%
maximum binding) to gpl2Oeither alone or competed with Fabs L39, L40, L78 or L41 at a concentration 10 times (B)or 100
times (Ed) that giving 75% maximum binding in previous titration experiments. c) Binding of mouse anti-V2 Ab 684-238 (at a
concentration giving 75% maximum binding) to gp120 either alone or competed with Fabs L39, L40, L78 or L41 at a concentration 10 times @) or 100 times) . ( that giving 75% maximum binding in previous titration experiments. d ) Binding of
whole human lgGl b12 (at a concentration giving 75% maximum binding) to gpl20 either alone or competed with Fabs L78
(60 pghnl), b12 (25 pghnl), SC258 or 684-238 at a concentration 100 times that giving 75% maximum binding in previous
titration experiments.
are that theformeris
affected by severalamino acid
changes in addition to those affecting L28 and LA2 and has
a somewhat higher affinity for gp120. However, the current data do not clearly explain why L41 was only retrieved after masking.
To further explore the nature of the novel epitope, studies on the effects of gp120 glycosylation and conformation
on Fabbindingwereconducted.Glycans
represent approximately 50% of the total m.w. of gp120 and seem to
play an important role in reducing the efficacy of the humoral immune response to viruses, e.g., by masking neutralizing sites (64-66). The epitopes of all the CD4bs Abs
were greatly affected by changes in glycosylation, indicating eitherthat (pl-4)-linked galactose from N-linked sugars is included in the binding site orthat the carbohydrates
Table IV. Neutralization of HIV-1 by recombinant human Fab
fragments as measured by microplaque assay"
Neutralization Titer
Fab
L28
L33
L42
L52
L39
L40
L4 1
L78
b12
MN
(pdml)
1a1
(pdml)
4.2
>40
0.7
>50
1.1
4 (59%)
0.9
>50
>50
>SO
0.8
1.6
0.7
>50
40.8
8.2
2 .o
0.1
a The Ab neutralization titer is expressed as the concentration (pLp/ml)of
Fab required to inhibit plaque formation to 50%.
904
NEUTRALIZING Abs
HUMAN HIV-1
FROM PHAGE LIBRARIES
are responsible for keepingthetertiarystructure
of this
HIV-1donor libraryandreportedpreviouslyas
s7 (11,
region of the molecule intact. However, the Abs are not
37). Only three amino acids were different in the heavy
directed to carbohydrate alone, inasmuch as they did not
chainvariableregion. The reasonforthiscould
be that
react with reduced but fully glycosylated gp120.
In conthese sequences are conserved or, more likely, that the L
trast with CD4bs Abs, binding of the three V2-dependent
library was cross-contaminated during construction with
Abs was not, or only slightly, affected by the enzymatic
phage from theotherlibrary. We have found that such
treatment, indicating that these carbohydrates are not incross-contamination can be a complicating factor in recluded in their binding site.
trieving Abs against a given epitope from libraries conIt has been reported that most broadly neutralizing hustructed from different patients. However, thepossible
man Abs are directedagainstdiscontinuous epitopes on
phage contamination, if any, does not interfere with the
gp120 (67). All Abs obtained from this HIV-1 donor liconclusions reported in this paper. Rather, the described
brary seem to recognize such discontinuous epitopes, as
masking method may be a useful strategy to avoid rebinding of all the Abs was affected by gp120 denaturation.
trieval of knowncontaminating phageIAbspecificities
There were, however, differences in the extent to which
from libraries.
binding was affected. Whereas binding of all Abs to the
In conclusion, themaskingproceduredescribed
is
CD4bs was completely abolished, the three V2-dependent
shown to be a valuable approach to retrieve Abs reactive
Abs were still able tobind appreciably to denatured gp120,
with moreminor epitopes,asdemonstrated
by the sucalthough apparently with much reduced affinity as comcessful isolation of human mAbs to a novel neutralizing
pared with intact gp120.
conformational epitopeinfluencedbythe
V2 andthe
Several of the V2-dependent murine mAbsdescribed in
CD4bs regions. Because Fab L78 is fairly broadly and pothe literature are fairly broadly neutralizing,suggesting the
tently neutralizing, it may be auseful component of a mixture
epitopes recognized are relatively conserved across strains
of anti-HIV-1 human mAbs for passive immunotherapy.
(14, 15). One of the VZdependent Fab (L78) showed efficient neutralization of both the MN and LA1 strains of
Acknowledgments
HIV-1, as measured by a plaque reductionassay using
We are particularly grateful to the donors for their cooperation, and to
MT-2 target cells. Against both strains, this Fab exhibited
Clifford Lane and Robert Walker (NIAID, National Institutes of Health)
50% neutralizationatapproximately2Fg/ml.Using
an
for kindly providing clinical samples used in this study. We thank Glenn
Pilkington, Roman Rozenshteyn, Anthony Williamson, Terri Jones, and
envelope complementationassay, L78 exhibited 50% neuShu-Wing Poon for their contributions to this work. We express gratitude
tralization at about 10 Fg/ml for the MN strain HIV-1. Of
to Jim Chambers for sequencing. The following reagents were obtained
the retrieved anti-CD4bs Fabs, L42 in particular showed
through the AIDS Research and Reference Reagent Program, Division of
potentneutralizationagainstthe
two strains,with 50%
AIDS, NIAID, National Institutes of Health: mAb 1IIB-V3-13 from
neutralization at less than 1 &ml in both assays. The
Dr. Jon Laman; mAb V3 (RN3-50.1) from Repligen Corporation; mAb
Q425 from Drs. Peter Kwong and Dr. Quentin Sattentau; and soluble
ability of L78 and L42 to neutralize primary isolates of
HIV-1 will be assessed after theirconversion to whole Abs recombinant CD4 from Dr. Ray Sweet.
(68). The neutralizing efficacy of the Fab fragments may
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