Identification and characterization of a neutralization site within the second variable region of human immunodeficiency virus type 1 gp120.

Copyright © 1992,AmericanSociety forMicrobiology

Identification and

Characterization of

a

Neutralization Site within

the Second

Variable

Region

of Human

Immunodeficiency

Virus

Type 1

gpl20

MICHAEL S. C. FUNG,1* CECILY R. Y.

SUN,'

WAYNE L.

GORDON,'

RUEY-SHYAN

LIOU,1

TSE WEN

CHANG,'

WILLIAM N. C.

SUN,'

ERIC S. DAAR,2AND DAVID D. HO3 TanoxBiosystems, Inc., 10301 Stella Link, Houston, Texas

770251;

Division ofInfectious Diseases,

Cedars-Sinai Medical Center, University of California at LosAngeles, Schoolof Medicine, LosAngeles, California 900482; and Aaron Diamond AIDS Research Center,

New York University School of Medicine, New York, New York

100163

Received 13 August 1991/Accepted 31 October 1991

Twomonoclonal antibodiesdesignated BAT085 and G3-136 were raised by immunizing BALB/c mice with gpl20purified from humanimmunodeficiencyvirustype1(HIV-1)IIIB-infected H9 cell extracts.Among three HIV-1 laboratory isolates (IIIB, MN, and RF), BAT085 neutralized only IIIB infection ofCEM-SS cells, whereasG3-136 neutralized both IIIB and RF. These antibodies also neutralized a few primaryHIV-1isolates inthe infection ofactivated human peripheral blood mononuclear cells.Inindirect immunofluorescenceassays, BAT085 bound to H9 cells infected with IIIB or MN, while G3-136 bound to H9 cells infected with IIIB orRF, but not MN.Usingsequence-overlapping synthetic peptidesof HIV-1 IIIBgpl20, thebinding site of BAT085 and G3-136 wasmapped to apeptidicsegment in the V2 region(amino acid residues 169 to 183). The binding of these antibodies to immobilized gpl20 was not inhibited by the antibodies directed to the principal neutralization determinant in the V3 region or to the CD4-binding domain of gpl20. In a competition enzyme-linkedimmunosorbent assay, soluble CD4 inhibited G3-136 but not BAT085 from binding togpl20. Deglycosylation of gpl20 by

endo-o-N-acetylglucosaminidase

H or reduction of gpl20 by dithiothreitol diminished its reactivity with G3-136 but not with BAT085. These results indicate that the V2 region of gpl20 contains multiple neutralization determinants recognized by antibodies in both a conformation-dependent and -inconformation-dependent manner.

Intensive efforts in AIDS research have been focused on the development ofan effective vaccine for protection of individualsatrisk for humanimmunodeficiencyvirus type 1 (HIV-1)infection(4, 45). It has becomeevidentthathumoral responses play crucial rolesin protective immunity against infection byHIV-1 and itsprogression to disease states. In thisrespect, Wendleretal. (49) showedthathigher neutral-izingantibody titers correlated with better prognosisand low or decreasing neutralizing antibody titers correlated with diseaseprogression. Jacksonetal.(19) and Karpas etal. (22, 23) demonstrated that AIDS-related complex and AIDS patients treated with hyperimmune plasma from asympto-matic HIV-1-seropositive individuals showed clinical im-provement, which correlated with reduction in HIV-1 vire-mia. More recently, preliminary studies by Emini et al. (7) showed that chimpanzees injected with a mouse-human chimeric HIV-1-neutralizing monoclonal antibody (MAb) were protected from infection by the virus.

The envelope proteins of HIV-1, gp120 and gp4l, are knowntobe the major targets forHIV-1-neutralizingactivity inthe seraofHIV-1-infectedindividuals (13, 18, 33, 48) and of animals immunized with the viral proteins (3, 28, 40). Several neutralization sites have been identified in

gpi20,

including an immunodominant epitope in the V3 region (amino acid residues 308 to 322), commonly known as the

principal

neutralization determinant(PND) (9, 21, 24, 27, 32, 43, 44). Antibodies raised against this continuous epitope

*Correspondingauthor.

usually exhibit

type-specific

neutralizing activity

against

the homologous viral isolates (11, 12, 33, 40, 43). However, recentstudies byJavaherianet al. (20) andWhite-Scharfet al. (50)showed thatantibodies raised inanimals againstthe relatively conserved

portion

ofthe PND displayed broad-ened reactivity with multiple HIV-1 isolates. Anti-PND antibodies do not block virus

binding

to CD4 but prevent virus internalization subsequent to CD4 binding (31, 44). Anothercontinuousneutralization siteongpl20is located in the C4 region (amino acid residues 413 to 447), which is found to be involved in CD4-gpl20 interaction (29, 47). Antibodies tothis region exhibit broad neutralizing activity against divergentHIV-1 isolates, but this neutralization site appears to be ratherimmunosilentinHIV-1-infected individ-uals(47).

Sera from most HIV-1-infected individuals usually have low levelsof broad neutralizingactivityagainst HIV-1 (2, 13, 48), which were found to be associated with antibodies directed to conformational or discontinuous epitopes on gpl20 (13). Consistentwiththis finding, we havereporteda human MAb

(I5e)

which recognizes adiscontinuousepitope ongpl20 involved in CD4binding andin antibody neutrali-zationofmultiple HIV-1 isolates (16). We also generated a HIV-1-neutralizing murine MAb

(G3-4),

which identified anotherdistinctconformational epitopeon gpl20involved in theinteraction with CD4(15).

Inthisreport, wedescribe thegeneration and characteri-zationoftwomurine MAbsdesignatedBAT085andG3-136 which defineaunique neutralization domain in the V2 region ofgpl20.

848

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MATERIALSAND METHODS

MAb reagents and polyclonal antibodies. Murine HIV-neutralizing MAb BAT123 immunoglobulinGl(K) [IgGl(K)] bindstothe PND inthe V3 region ofIIIB

gpl20

(amino acid residues 308 to 322) (32). Murine MAb G3-519

[IgGl(K)]

recognizesa component of the CD4-binding domain in the C4

region

ofgpl20 (amino acid residues 423 to 437) (47). Murine MAb G3-4 [IgG2a(K)] recognizes a conformational neutralizing epitope on HIV-1gp120(15).

I5e

[IgGl(K)] is a

HIV-1-neutralizing

human MAb (a gift from J.

Robinson,

Louisiana State University School of Medicine, New

Or-leans)

which also recognizes a distinct conformational

epitope

on gpl20 (16). The murine MAbs were produced fromascitic fluid of BALB/c mice(Harlan Sprague-Dawley, Inc., Houston, Tex.) injected with the hybridomas and

affinity

purified by using recombinant protein A-Sepharose

(Repligen,

Boston, Mass.). I5e was produced from culture supernatant of the hybridoma and purified by protein A

affinity chromatography.

Polyclonal anti-HIV-1

gpl20

anti-bodies (D6205) (International Enzymes, Inc., Fallbrook,

Calif.)

were

generated by immunizing

sheep withasynthetic

peptide encompassing

a

highly

conservedpeptidic segment

(APTKAKRRVVQREKR)

in the carboxy terminus of HIV-1(BH-10

strain)

gpl20

(amino acid residues 497to511)

(35-37).

The antibodieswereisolated from the sheep hyper-immune sera

by affinity

chromatography usingthe specified

synthetic peptide coupled

to

Sepharose.

Cells and HIV-1 isolates. CEM-SS

cells,

a

syncytium-sensitive clone of CEM cells (a

gift

from P. Nara, National Cancer

Institute),

were maintained in RPMI 1640 medium

(GIBCO Laboratories,

Grand

Island,

N.Y.)

supplemented

with

10%

heat-inactivated fetal bovine serum(GIBCO), 100 Uof

penicillin

per

ml,

and100

p.g

of

streptomycin

per ml.H9 cells

chronically

infected withHIV-1IIIB, MN,orRF,were maintained inRPMI 1640medium with15% heat-inactivated fetal bovine serum.These infected cells were derivedfrom

single-cell

clones which were tested to be almost 100%

expressing

gpl20

on the cell surface

by

immunofluorocyto-metric methods. The culturesupernatants fromthese HIV-1-infected cells

containing high

reverse

transcriptase

activi-ties were

kept

at -

160°C

and used forHIV neutralization assays.

Peripheral

blood mononuclear cells

(PBMCs)

were isolated from

HIV-1-seronegative

donors

by

Ficoll-Paque

density

gradient centrifugation,

and were stimulated for 3

days

with 2

jig

of

phytohemagglutinin (PHA) (Sigma

Chem-ical

Co.,

St.

Louis,

Mo.)

per ml in RPMI 1640 medium

supplemented

with15% heat-inactivated fetal bovineserum, 10% human interleukin-2

(Cellular Products, Inc.,

Buffalo,

N.Y.),

100 Uof

penicillin

per

ml,

and100 ,ugof

streptomycin

perml. After

stimulation,

PBMCs were washed and main-tained in the

complete

growth

mediumfor

infectivity

assays.

Primary

HIV-1isolates

(LS,

RP,

CC,

LL,

TB,

and

AC)

were

obtained from

plasma

specimens

from

HIV-1-seropositive

individuals,

as

previously

described

(5, 16,

17).

TheJR-CSF isolate was

kindly

provided by

I. Chen

(University

of California at Los

Angeles,

School of

Medicine,

Los

Ange-les).

Generation and

screening

of MAbs

against

HIV-1

gpl20.

Affinity-purified

HIV-1 IIIB

gpl20

was used to immunize maleBALB/c mice. Our standard

procedures

for

purification

of

gpl20

from infected H9 cell

lysates,

immunization of

mice,

and

generation

of

hybridomas

were

previously

de-scribed in detail

(10, 47).

Briefly,

3

days

after the final

immunization,

the mice were

sacrificed,

and

spleen

cells were isolated and fused with

Sp2/0 myeloma

cells. After

selection using hypoxanthine-aminopterin-thymidine (HAT) medium, culture supernatantsfrom the wells of microdilu-tionplates with hybridomas were tested for reactivity with gpl20as coating antigen by enzyme-linked immunosorbent assays (ELISAs). Culture supernatants showing strong gpl20 reactivity were tested for staining live HIV-1 IIIB-infected cells by an indirect immunofluorescence method (47). Hybrids fromselected fusion wells were expanded and cloned. MAbsfromhybridomas selected for further charac-terizationwere produced in mouse ascitic fluid and purified by proteinA affinitychromatography (47).

gp120 ELISA for screening of hybridomas. The procedures were similar to those described earlier (10, 47). Wells of Immulon 2 microdilution plates (Dynatech, Chantilly, Va.) were coated overnight at room temperature with 100 ,ul of purified gpl20 (0.1

p.g/ml)

in phosphate-buffered saline (PBS). The wells were then treated with 5% BLOTTO in PBS for 1 h at room temperature and washed with PBS containing 0.05% Tween 20 (PBST). Next, 100 ,ul oftest culturesupernatant wasaddedtoeach well for1 h at room temperature.The wellswerethenwashed andincubated for 1h at room temperaturewithhorseradish peroxidase-conju-gatedgoatanti-mouse IgG (Jackson ImmunoResearch Lab-oratories, Inc., West Grove, Pa.) diluted in PBST containing 1% bovine serumalbumin(PBSTB). After anotherwashing step, boundantibodiesweredetected by reaction with0.1% tetramethyl benzidine and 0.0003% hydrogen peroxide as substrates. The optical density at 450 nm (OD450) of the reaction solutionwas read with anELISA reader.

Neutralization assays of HIV-1 laboratory isolates using CEM-SS cells. The

syncytium-forming

microassays using CEM-SScells were

performed

as

previously

described (10, 39).

Briefly,

50 ,ul of diluted MAb was mixed with50 ,ulof viral culture supernatantcontaining200 syncytium-forming units(SFU) ofHIV-1 IIIB, MN,orRFand incubated for1 h at room temperature. The mixtures were added into microculture wells

containing

5 x 104 DEAE-dextran-treated CEM-SS

cells,

andthecellculturesweremaintained in 5%

CO2

at37°C for 3 to 4days. The

syncytia

were then enumerated underaninverted

microscope.

The

neutralizing

activity

was

expressed

as

ID50,

definedasthe concentration

required

to achieve 50% inhibition of the infection

(i.e.,

Vn/Vo =

50%),

where Vn is the numberof SFU inthetest wells andVo isthe number of SFU in the control without testantibodies. In the

study

ofthe

epitope

specificity using

peptide

V1SP, the

neutralizing

activity

ofBAT085 at 12.5

,ug/ml

(ID50) was tested for inhibition

by

incubating

the

antibody

with various concentrations of the peptidefor 20 min at room temperature,

prior

to the addition of a viral inoculumof100SFUs ofHIV-1IIIB.Theirrelevant

peptide

a-endorphin (Peninsula

Laboratories, Belmont, Calif.)

was usedforacontrol.

Neutralizationassays ofprimaryHIV-1isolates

using

PHA-activated PBMCs. The titration of

primary

HIV-1 isolates and their neutralization assays were

performed by

our

standard

procedures (5, 17).

An inoculum of 50 50% tissue culture infective doses wasincubated with various

concen-trationsof MAbs

(0.01

to25

,ug)

in 1 ml of final culture for 30 minat

37°C.

ThecontrolcontainednoMAb. On

day

4to

7,

depending

onthe

replication

kinetics of the

primary

HIV-1 isolates,the HIV-1

p24

antigen

levels of the

antibody-treated

and untreated cell cultures were measured with Abbott HIV-1

Antigen

Kits

(Abbott

Laboratories,

North

Chicago,

Ill.).The

neutralizing

activity

ofthe

antibody

is

expressed

as thepercent reduction of

p24

antigen

levels in the

antibody-treated wells

compared

with that of the control.

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Flow

cytometry.

The

binding

of

BAT085

and G3-136toH9 cellsinfected withHIV-1

IIIB, MN,

orRFwas

analyzed

by

flow

cytometric

methods

using

aCoulter EPIC cell

analyzer

(Coulter

Electronics, Hialeah,

Fla.)

as

previously

described

(25).

The bound MAbs were detected

by

fluorescein-conju-gated

goat

anti-mouse

IgG (Jackson

ImmunoResearch

Lab-oratories, Inc.).

Generation of HIV-1

gpl20

peptides.

All

peptides

used in

epitope mapping

were

synthesized by

using

the RaMPs

peptide

synthesis

system

(Du

Pont

Co.,

Wilmington,

Del.)

by

the

9-fluorenylmethoxycarbonyl synthesis

protocol

de-scribed in the manual

(6a).

The

purity

of the

RaMPS-synthesized

peptides

was characterized

by

high-perfor-mance

liquid

chromatography,

and the

predicted

structure

wasassessed

by

fast-atom bombardmentmass

spectrometry

analysis.

Amino acid sequencesof the

synthetic

peptides

for

IIIB

(HXB2

clone),

MN,

and RF isolates ofHIV-1 were

obtainedfrom

Myers

etal.

(38).

Epitope mapping

using synthetic

HIV-1

gpl20 peptides.

Preliminary epitope mapping

was

performed by using

nitro-cellulose

strips impregnated

with

sequence-overlapping

syn-thetic

oligopeptides

(9

to15amino acid

residues)

encompass-ing

the entire

length

ofHIV-1

IIIB

gpl20

polypeptide

chain

(24)

(from

S.

Petteway

formerly

ofDuPont

Co.).

Theamino acid sequenceswerederived from HIV-1 clone HXB2

(38).

The reactivities of anti-HIV-1

gpl20

MAbs with these pep-tidesweredetermined

by

the methods

previously

described

(32).

Briefly,

the nitrocellulose

strips

were

incubated

over-night

atroom

temperature

withtestMAbsat10

,ug/ml

in5% BLOTTO. The

strips

were then washed with PBST. The

bound

antibody

was thendetected

by

incubating

the

strips

with diluted horseradish

peroxidase-conjugated

goat

anti-mouse

IgG

(Jackson

ImmunoResearch Laboratories,

Inc.)

for1hatroom

temperature.

The

strips

were

washed,

andthe

color reaction was

developed

with a substrate solution

containing

0.3%

4-chloro-1-naphthol

and 0.005%

hydrogen

peroxide.

Further fine

epitope mapping

was

performed by peptide

ELISA. Wells of Immulon 2 microtest

plates

were coated

overnight

at room

temperature

with 100

,ul

of the

synthetic

peptides

(1

,ug/ml)

in PBS. The wells werethentreatedwith

5%

BLOTTO

for 1 h atroom

temperature.

After the wells were washed with

PBST,

100

pAl

of

BAT085

or G3-136

(1

jig/ml)

was addedto the wells for reaction for 1 hat room

temperature.

The wells were then

washed,

and the bound

MAb was then detected

by

horseradish

peroxidase-conju-gated

goat

anti-mouse

IgG.

The colorreaction was

devel-oped

by

incubation

with the

peroxidase

substrate solutionas

described above.

Reactivity

was

regarded

as

positive

if the

OD450

wastwofold

higher

thanthat ofthe wellscoatedwith the

irrelevant

peptide

a-endorphin.

Competition

ELISA. The effects of

anti-HIV-1

gpl20

MAbs,

peptide

V15P

and recombinant solubleCD4

(sCD4)

on the

binding

of

BAT085

and G3-136to HIV-1 IIIB

gpl20

were determined

by

the

procedures

previously

described

(35-37).

sCD4wasobtained from

Biogen,

Inc.,

Cambridge,

Mass. Wells of Immulon 2 microtest

plates

were coated

overnight

at room

temperature

with 50 ,uof a solution

containing

5

,ug

of

affinity-purified sheep

anti-HIV-1

gpl20

(D6205,

International

Enzymes,

Inc.)

per ml.The wellswere

then treatedwith5%

BLOTTO

for1hatroomtemperature.

Fifty

microliters

of

purified gpl20

(0.5

pug/ml)

wasthenadded toeach wellfor

incubation

for2hatroom

temperature.

The

plates

werethen washed.Onehundred

microliters

ofdiluted

biotinylated

MAb was added to the wells with or without

various amounts of

competing

MAbs

(including

BAT085,

G3-136,

G3-4, BAT123, G3-519, and ISe), peptide V15P or sCD4. The biotinylated MAbswereused at dilutions which gave half-maximalbindingto thecaptured

gpl20.

The incu-bationtimewas 1 h at room temperature. The

plates

were washed and reactedwith 100 Ll of horseradish

peroxidase-conjugatedstreptavidin(diluted1:2,000 inPBSTB) (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room tem-perature.Theplateswerewashed

again

and allowed to react with the peroxidase substrate solution as described above. The degree of inhibition was

expressed

as the

percent

decrease in

OD450

in the test wells

compared

with the OD of control wells without the inhibitors.

RIPA. The radioimmunoprecipitation (RIPA)

procedure

wascarried out as described earlier(34, 47).

Briefly,

HIV-1

IIIB-infected

H9 cellsweremetabolicallylabeled for 4 h with

[35S]cysteine

and[35S]methionine(100

,uCi/ml;

ICN

Pharma-ceuticals, Inc., Irvine,Calif.)and

suspended

in RIPA

lysing

buffer (50 mM Trishydrochloride

[pH

8.0], 150 mMNaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl

fluoride).

To examine the effect ofantibody

binding

to

gpl60

and

gpl20

after reduction of disulfide linkages, the metabolically la-beled cell lysates was treated with 0.1 M dithiothreitol in PBS at 37°C for 1 h. Dithiothreitol was then removed

by

dialysis.The effect ofdeglycosylationof

gpl60

and

gpl20

on

antibody binding was also examined by treating the cell lysates with 100 mU of

endo-o-N-acetylglucosaminidase

H for 2.5 h at 37°C. The treated or untreated

lysates

were

preclearedwithprotein A-Sepharosebound to rabbit antise-rum to mouse kappa light chain

(K-PAS)

for 3 h at room

temperature.RIPA wasperformedby adding3

,ug

of

purified

MAb (BAT085,

G3-136,

or G3-4) and 0.2 ml of a 10% suspension of K-PAS to 200 ,ulof labeled and clarified

lysate.

The sampleswere incubatedfor 18 h at4°C,and the beads were washed with RIPA lysing buffer. The pellets were

suspended inelectrophoresis samplebuffer and boiled for 3

min.

Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,followedby

autoradiog-raphy.

RESULTS

Generation and characterization of MAbs BAT085 and G3-136. Affinity-purified gpl20 prepared from lysates of HIV-1 IIIB-infected H9 cells was used to immunize mice. The culturesupernatantsofhybridsweretestedfor reactiv-ity with

gpl20

by ELISA. Antibodies exhibiting strong

gpl20

reactivity

were tested forstaining live HIV-1 IIIB-infected H9 cellsbyanindirect immunofluorescence method inorder to determine whetherthe antibodies reacted withgpl20on

infected cells. The MAbs from cloned hybrids were tested for neutralizing activity against HIV-1 IIIB using a syncy-tium-forming assay. Two neutralizing MAbs

designated

BAT085 and G3-136 [both IgGl(K)] were identified for fur-thercharacterization.

In the flow

cytometric

assayshown in Fig. 1A,BAT085at 10

p.g/ml

stained 87 and 35% of H9cellsinfected withHIV-1 IIIB and

MN,

respectively.

It didnotstainH9cells

infected

with HIV-1 RF. G3-136 stained 73 and 91% of H9 cells infected with HIV-1 IIIB andRF, respectively (Fig. 1B)but did not stain H9 cells infectedwithHIV-1 MN.

In the in vitro neutralizationassaysagainstHIV-1

labora-tory

isolates

using

CEM-SS cells, BAT085neutralized IIIB with

ID50

of 12.5

p,g/ml

(Fig.

2A). Itdid not neutralize MN and RF. G3-136 neutralized IIIB andRF with ID50 of 16 and

2

pg/ml, respectively

(Fig. 3A).

It did not neutralize MN.

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http://jvi.asm.org/

1.00

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FIG. 1. Bindingactivities of BAT085(A)andof G3-136(B)toH9 cells infected with HIV-1 IIIB ( *), MN (....), or RF ( .Uninfected H9 cells( )were used foracontrol. The H9 cells(107cellsperml)were incubated withorwithout the antibodiesfor 30 minonice. After the cellswerewashed,theywere

incubated with goat anti-mouse IgGcoupled withfluorescein iso-thiocyanatefor 30min, washed,andfixed withparaformaldehyde. Fluorescenceintensitywasanalyzedbyflowcytometry.

Neutralizing

activities

against

the

infectivity

ofseveral

pri-mary HIV-1 isolates were also tested

by using

PHA-acti-vated PBMCs. BAT085neutralized two

(LS

and

RP)

of six isolates tested with an

ID50

of lessthan 1

,ug/ml (Fig.

2B);

G3-136neutralizedthree

(AC, LS,

and

TB)

ofsevenisolates

with

ID50 ranging

from0.03 to0.4

,ug/ml

(Fig. 3B).

Epitope mapping

ofBAT085andG3-136. Todelineate the

epitopes

on

gpl20

that these twoMAbs

recognize,

we first

tested theeffect of other

previously

characterized

neutraliz-ing

anti-HIV-1

gpl20

MAbs on the

binding

of

biotinylated

BAT085 and G3-136 to

gp120.

These other MAbs include

BAT123,

G3-519, G3-4,

and

ISe;

the lasttwo MAbs recog-nizedistinct conformational

neutralizing

epitopes

on

gp120.

In the

competition

ELISA,

purified

HIV-1 IIIB

gpl20

was

captured

by

affinity-purified

sheep

anti-HIV-1

gpl20

anti-bodies which are

specific

for a

highly

conserved

peptidic

segmentin the

carboxy

terminusof

gp120.

The immobiliza-tion of

gpl20

didnothinderthe

binding

of

BAT123, G3-519,

G3-4, I5e, BAT085,

and G3-136.

Figure

4A shows that the

binding

of

biotinylated

BAT085to

gpl20

was

partially

inhib-ited

by

G3-4

(80%)

and G3-136

(65%)

at 40

,g/ml.

In contrast,

BAT123, G3-519,

and ISe hadnoeffect.

Unconju-gated

BAT085 at 40

,ug/ml

completely

abolished

binding.

When the

binding

of

biotinylated

G3-136 to

gpl20

was

tested,

G3-4inhibited the

binding

as

effectively

asG3-136did

(Fig. 4B). However,

BAT085 inhibitedthe

binding by only

33%evenat40

,ug/ml,

and

BAT123,

G3-519,

andISe hadno

effect. These results were confirmed

by

examining

the

ef-0

0.10

0.01

c 0

0

0

()

04.

100

80

60

40

20

0

10 BAT085, sg/mI

100

0 0.01 0.10 1.00 10.00 100.00

BAT085,sg/ml

FIG. 2. Neutralization ofHIV-1 by BAT085. (A) Inhibition of infection ofCEM-SScellsby laboratory HIV-1 isolateIIIB (0), MN (0),and RF(A).Aviral inoculumof100SFUwasusedtoinfect the cells in the presence or absence of various concentrations of

BAT085. Vnisthemeannumber of syncytia in duplicatetestwells, and Vo is the mean number of syncytia in the duplicate control wells. (B) Inhibition of infection ofPHA-activated PBMCs from HIV-1-seronegative donorsby primaryHIV-1 isolates LS(0), RP (V), CC(O), JR-CSF(O),LL(A),andTB(A). Aviralinoculum of50 50% tissuecultureinfective doseswasusedtoinfect the cells in the

presence or absence of various concentrations of BAT085. The neutralizing activity of the antibody is expressed as the percent

reductionofp24 antigenlevels in the antibody-treated wells

com-paredwiththatof the control.

fects of BAT085 andG3-136 on the binding of biotinylated

G3-4, BAT123, G3-519, and ISe to gpl20. As expected, BAT085 and G3-136 competed with G3-4 but not with BAT123, G3-519, and ISe (16; also unpublished results).

To define thebinding epitope(s) of BAT085 and G3-136on

gp120, the MAbs were examined by using nitrocellulose

stripsimpregnatedwithsequence-overlapping synthetic

pep-tides encompassing the entire length of HIV-1 IIIB gpl20 polypeptide chain. It was found that BAT085 and G3-136

reactedspecificallywithapeptidicsegment, VQKEYAFFY

KLDIIP (referred to as V15P) in the V2 region ofgp120, amino acid residues 169to183. To confirm thisobservation, overlapping oligopeptides encompassing the peptidic

seg-ment (amino acid residues 159 to 198) inHIV-1 IIIB gp120 (Table 1) were synthesized for ELISAs and liquid-phase

competition assays. When these peptides were usedas the solid-phase antigens at 1 ,ug/ml, BAT085 reacted strongly withV15P(OD450 = 1.4), andweakly with L20T(OD450 =

0.13). It did not reactwith F1SY, V1OK, and AlOP, giving OD450s less than twofold greater than the negative control a-endorphin(OD450= 0.01). G3-136also reacted withV15P

A

.8

--..P--- - -0

1

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1.0(

0.14

0.0

100 80

60

40

20

0

) 0

1 ... ~.

1 10 100

G3-136, yg/ml

B

*

a

0 0.01 0.10 1.00 10.00 100.00

G3-136. /g/ml

FIG. 3. Neutralization of HIV-1 by G3-136. (A) Inhibition of

infection of CEM-SS cells by laboratory HIV-1 isolates IIIB (0), MN(0),and RF(A). (B)Inhibition of infection of PHA-activated PBMCs fromHIV-1-seronegativedonorsby primaryHIV-1 isolates AC(0),LS(0),LL(A),TB(A),CC(O),JR-CSF(O),and RF(V). The neutralizing activities of the antibody were determined as

described in thelegendtoFig.2.

(OD450 = 0.44) but did not reactwith F15Y, L20T, V1OK,

and A1OP. In the determination ofreactivity with different HIV-1isolates,BAT085 didnot reactwithpeptides M15S and T15P, which correspond tothesame region of peptide V15P

100

c

.0

-C_c

-0.

0

0

L-80 60 40 20

0 0.1

100

.0

*t_

.0

a-80

60

40

20

0

1.0 10.0

Competing antibodies, ug/ml

0 0.1 1.0 10.0

Competing antibodies, p,g/ml

FIG. 4. Inhibition of thebindingofbiotinylatedBAT085(A)and biotinylatedG3-136(B)tocapturedHIV-1 IIIBgpl20in ELISAby BAT085 (A),G3-136(0),G3-4(0), BAT123(A), G3-519(l),and

15e(0).ThecompetitionELISAprocedureis described inMaterials and Methods.

in the gpl20 of HIV-1 MN and RF, respectively. G3-136 reacted with T15P(OD450 = 0.23), butnotwithM15S.

Inthe liquid-phase competition bindingassay examining

theeffects ofpeptidesonthebinding of biotinylated BAT085

[image:5.612.319.559.63.375.2]

tocapturedgpl20, V15P inhibited the binding of BAT085to

TABLE 1. Epitopemapping of BAT085 and G3-136 to theV2region of HIV-1gpl20by peptide ELISA

Viralpeptide Sequence (amino acids

159-198")

Reactivity(OD450)

andantigen BAT085 G3-136

HIV HXB2

160 170 180 190

F15Y FNISTSIRGKVQKEY 0.01 0.03

V15P VQKEYAFFYKLDIIP 1.40 0.44

L20T LDIIPIDNDTTSYSLTSCNT 0.13 0.02

V1OK VQKEYAFFYK 0.02 0.01

AlOP AFFYKLDIIP 0.03 0.01

HIV MN

M15S M----LL----VS 0.01 0.02

HIV RF

T15P T--K--L----VV- 0.01 0.23

aDeduced amino acid sequences are obtained from Myers et al. (38). Homologous amino acids are represented by dashes. TheOD450of the blank wasbetween 0.01 and 0.03.

0

c

c 0

c 0

0

C)

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100

c

0

.0 .r_

-c

._-c

0) 0-0)

80 60

40

20

0.01 0.10 1.00 10.00

Competing peptides, tg/ml

FIG. 5. Competitionof thebinding between biotinylated BAT085 andcapturedHIV-1IIIB gpl20 by the epitopepeptide V15P (0) and

theirrelevant peptidea-endorphin(0).Theamino acidsequenceof

V15P is shown in Table 1. The ELISA procedure is described in

Materials and Methods.

gp120inadose-dependentmanner(Fig.5).Thea-endorphin

peptide had no effect, evenataconcentrationof 40 p.g/ml.

Thebinding specificity ofV15PtoBAT085 was also

exam-inedintheneutralizationassayofHIV-1 IIIBusing CEM-SS cells.Figure 6 shows that V15P exhibitedadose-dependent

inhibition of the neutralizing activity of BAT085, while a-endorphin hadnoeffect. Either peptide alone didnotaffect theinfectivity of HIV-1 IIIB.

Effectsof sCD4 on thebinding ofBAT085 and G3-136 to

gpl2O. Toexaminewhether the neutralizing epitope mapped to the V2 region was involved in the interaction between

CD4 and gpl20, the effect of sCD4 on the binding of the

biotinylated MAbs to captured gp120 was tested. sCD4

inhibitedthe binding of G3-136, G3-4, and G3-519 togpl20 (Fig. 7), althoughnotcompletelyevenataconcentrationas

high as 40 ,ug/ml. The binding of BAT085 and BAT123 to gpl20 isnotaffected by sCD4. These resultssuggestthatthe

c

0

0

E

(0

-._-c

co

0

c

'

50

40

30

20

10

0

0 0.01 0.10 1.00 10.00

Competing peptides,ug/ml

FIG. 6. Effects ofoligopeptides on the neutralizing activity of

BAT085 against HIV-1 IIIB. Asdescribed in Materials and

Meth-ods, CEM-SS cells(5 x 104)were infectedwith 100 SFU of HIV-1

IIIBin thepresenceofBAT085at12.5,ug/ml equivalenttoID50 (°>)-Various concentrations of the epitope peptide V15P (0) or the

irrelevantpeptideoa-endorphin (0)weremixed with theantibodyas

described in Materials and Methods. The percent inhibition of

syncytium formation is defined as the percent decrease in the

number ofsyncytia in the test wells compared with that of the

control wellswithout theantibody.

70 60

c

50-0

z

40-.c 30-c

20

00

r~10

0

0.01 0.10 1.00 10.00

sCD4, /g/mI

FIG. 7. Inhibitoryactivity of sCD4onthebindingof biotinylated

MAbsto

ca,ptured

HIV-1 IIIBgpl20 in ELISA. Thesebiotinylated antibodies, including BAT085 (D), G3-136 (A), G3-4 (A), G3-519 (O), and BAT123 (0) were used at dilutions which gave

half-maximal bindingtothe capturedgpl20.Various concentrations of

sCD4 were mixed with the labeled antibodies for binding to the gpl20 for 1 hat room temperature asdescribed in Materials and

Methods.

epitopes recognized by BAT085 and G3-136 may be

dif-ferent, and that ofG3-136 isnear aCD4-binding domain of

gpl20.

Effect of reduction and deglycosylation of gpl20 on its

binding by BAT085 and G3-136. To study whether the binding of BAT085 and G3-136 to gp120 was affected by

conformational changes ofgpl20, the effects of reduction of disulfide linkages anddeglycosylation of gpl20were

exam-ined. As summarized in Table 2, the reactivity of G3-136 withgpl20 inaRIPAwasreduced when the virallysatewas

firstdenaturedbydithiothreitol.Whenaradiolabeled HIV-1

lysate was subjectedto

endo-p-N-acetylglucosaminidase

H digestion, the partially deglycosylated product of 90to 100 kDawasweaklyrecognizedbyG3-136 (Table 2). Inparallel

experiments, the reactivity of BAT085 with reduced or

deglycosylated gp120wasunaltered, whereas the reactivity

ofG3-4 with gp120 was completely abolished under these

conditions.

DISCUSSION

We reporthere thataunique neutralization domain inthe

V2region of HIV-1gp120 has been identified bytwomurine MAbs, BAT085 and G3-136, raised against HIV-1 IIIB gpl20. ThesetwoMAbs canneutralizeinvitro the infectiv-ity of certain laboratory and primary HIV-1 isolates. The immunologic and biologic properties of these MAbs are

summarizedinTable 2.

By usingsequence-overlapping synthetic peptides

encom-passing the entire length of the HIV-1 gpl20 polypeptide chain,thebindingsiteof BAT085 andG3-136wasmappedto thepeptidic segmentV15P(aminoacid residues 169 to183) in HIV-1 IIIB gp120 (Table 1). Confirming our findings,

Stephens et al. (46) recently reported that antisera to the

synthetic peptide definingthe corresponding segmentinthe

V2 region of HIV-1 SF2 gpl20(amino acidresidues 171 to 185) neutralized in vitro the infectivity of the virus. The

specificity ofpeptide V15Pfor BAT085hasbeenconfirmed bythe liquid-phase competitionassays asillustrated in Fig. 5 and6. The absence ofreactivity of these MAbs with the

shorterpeptides

(VlOK

and AlOP) ofthis regionindicates

-1'~~~~~~~~~~~,~

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TABLE 2. Properties of neutralizing MAbstothe V2 region of HIV-1gpl20a

Neutralization Staining cells Effectofdegly- Effect of disulfide Bindingtoepitope

MAb of HIV infectedwith HIV sCD4

inhibition

of cosylation on bond reduction on peptide(amino IIIB MN RF IIIB MN RF

gpl20

binding

gpl20

binding

gpl20

binding acids 169-183)

BAT085[IgGl(K)] + - - ++ + - - None None ++

G3-136 [IgGl(K)] + - ++ + - ++ + Reduced Reduced +

G3-4[IgG2a(K)] + - ++ + - ++ + Abolished Abolished

-aSymbols: +,reactive; -,unreactive; ++,stronglyreactive.

that the actual binding site may be composed of several amino acid residues interspersedwithin peptide V15P. The identities of the crucial amino acid residues remain to be elucidated. Despite the fact that both BAT085 and G3-136

can bind to peptide V15P, only G3-136 reacts with peptide T15P of HIV-1 RF andstrongly stainsH9cellsinfectedwith this viralisolate. It is still unclear which aminoacid residues inthesepeptides actually determine thebinding specificities of different HIV-1 isolates.

Inasmuch as the binding of G3-136 to gpl20 is partially sensitive to deglycosylation and reduction (Table 2), its binding epitope is likely to be conformation dependent. In contrast, thebinding of BAT085togpl20isnotsensitiveto conformational changes of gpl20. This subtle difference in the binding characteristics between G3-136 and BAT085 is consistent with the inability of BAT085 to compete effec-tively with the binding of G3-136 to gpl20 (Fig. 4B). We recently reported that a murine HIV-1-neutralizing MAb,

G3-4, recognized a conformational epitope in HIV-1 IIIB

[image:7.612.68.563.83.154.2]

gpl20 (Table 2) (15). Although G3-4 did not react with peptide V15P, it effectively inhibited the binding of G3-136 but not BAT085 to gpl20, suggesting that the binding epitopes ofG3-4 and G3-136 areoverlapping (Table 2). The

FIG. 8. Schematic representation of the epitopes for BAT085, G3-136, G3-4, G3-519, BAT123, andISe ongpl20and their spatial

relationship with one another and with the CD4-binding site as

determined by competitionELISA.

binding of G3-4 togpl20isstrictly conformation dependent, because its binding is completely abolished when gpl20is deglycosylated or reduced (15). On the basis of these find-ings, we conclude that the V2 region ofgpl20may contain multiple neutralization determinants constituted by either continuous or discontinuous structures.

Results from this study also indicate that the neutraliza-tion determinants in the V2 region recognized by BAT085 and G3-136 are likely to be topographically removed from the PND in the V3region and theCD4-bindingdomain inthe C4region, since thebinding ofBAT085 orG3-136 to gpl20 cannot be inhibited by BAT123 and G3-519 in ELISA. In addition, BAT085 and G3-136 do not inhibit the gpl20 binding of ISe, which recognizes a distinct conformational epitope yet to be defined (16). This observation provides credence to the possibility ofcombining HIV-1 MAbs and variousneutralizationepitopes in passiveimmunotherapyto conferimproved efficacy and broadened viralreactivity. In vitro experiments are in progress in our laboratories to address the functional interactions between these neutrali-zation sites.

Itisinteresting to notethatG3-136 neutralizes a heterol-ogous HIV-1 isolate (RF)moreefficientlythan the homolo-gous isolate(IIIB) (Fig. 3A),althoughits immunoreacitivity is stronger with peptide V15P of IIIB than with the corre-sponding peptideT15PofRF. Apossibleexplanation forthis observation is that the binding site on native HIV-1 IIIB gpl20 is not asaccessible as that ofHIV-1 RF. The acces-sibility oftheneutralization sitesongpl20canbeaffected by theintricate folding of gpl20,as well as the variable extentof glycosylation of gpl20 (8, 14, 30). The juxtaposition of discontinuous regions of gpl20 has been implicated in the association between gpl20and gp4l (26)and in the escape of HIV-1 variantsfromneutralizationbyantibodies(41,51). In linewiththis,recentstudiesby Davis et al. (6) indicated that glycosylation of gpl20 could affect the binding of anti-peptide antibodies to gpl20. Another possible explanation for the higher neutralizing activityof G3-136 against HIV-1 RFis thatsincethe binding ofG3-136 togpl20 ispartially conformation dependent (Table2),G3-136may fit the three-dimensionalconfigurationofthe binding site on thegpl20of the RFisolate better.

In order tounderstand the possible mechanisms of neu-tralization mediated by BAT085 and G3-136, we examined whethertheir binding to

gpl20

can affect

CD4-gpl20

inter-action. Indeed, likeG3-519, thebinding of G3-136 andG3-4 togpl20 can be inhibited by sCD4. In contrast, the binding ofBAT085togpl20is not affected in the presence of sCD4. Thesedatasuggests that the neutralizing activities ofG3-136 andG3-4maybemediated by interfering with the interaction between CD4 and gpl20, whereas BAT085 may have a distinct and as yet undefined mechanism. The inability of sCD4tocompletely abolish the binding ofG3-136,

G3-4,

or G3-519 to gpl20 can be explained by the fact that the

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structural determinants recognized by these antibodies are either only sterically proximal to the actual CD4 binding sites or are forming parts of the multiple discontinuous regions responsible for CD4 binding (1, 42). The spatial relationship of the newly identified V2 neutralization epitopes relative to the otherpreviously defined neutraliza-tion sites (V3, C4, and the I5e conformational determinant) and totheCD4-binding site isschematically showninFig.8. Onthe basisof thefindingsinthisstudy,weconcludethat the V2 region of gpl20 bears multiple and

overlapping

neutralization determinants. To better design active and passive immunization

strategies,

it is

important

to

study

whetherneutralizing antibodies tothisregion play anactive role in the protective immunity against HIV-1 infection in humans.

ACKNOWLEDGMENTS

We thankN. Chang foradvice, J.Robinson forantibody15e, S. Petteway forgp120 peptide-coatednitrocellulosestrips,N. Jonesfor technical assistance in peptide synthesis, and J. Wrightfor prepa-ration of the manuscript.

This study was supported in part by grants from the National Institutes of Health(AI25541,A128747, A127742,andA124030),the ErnstJungFoundation, and theAaron Diamond Foundation.

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