A monoclonal antibody to human immunodeficiency virus type 1 which mediates cellular cytotoxicity and neutralization.

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JOURNAL OFVIROLOGY, Feb. 1990,p.936-940 Vol. 64, No. 2 0022-538X/90/020936-05$02.00/0

CopyrightC)1990,AmericanSociety forMicrobiology

A

Monoclonal

Antibody

to

Human

Immunodeficiency

Virus

Type

1 Which Mediates Cellular

Cytotoxocity

and Neutralization

PER A.

BROLIDEN,l.2.3*

KRISTINA

LJUNGGREN,2'3

JORMA

HINKULA,1

ERLING

NORRBY,2

LENNART AKERBLOM,4AND BRITTA WAHREN1

DepartmentofVirology, NationalBacteriological Laboratory,l* Departments of Virology2and Immunology,3 Karolinska

Institute, Stockholm,

and

Department

of Veterinary

Microbiology, Biomedical Center,

Uppsala,4

Sweden

Received 5June1989/Accepted13October1989

Monoclonal antibodies(MAbs)wereraisedagainsthumanimmunodeficiencyvirustype1gpl20.OneMAb, P4/D10, was foundtomediatehighlyefficientantibody-dependentcellularcytotoxicityand virusneutralization. Thereactivitywaslocated to a

major

neutralizing region(aminoacids 304 to323)ongpl20.Five otherMAbs with a similarepitopicreactivity didnotshowanyantibody-dependentcellulancytotoxicity activitybut had a virus-neutralizingcapacity.

Understanding thebiological function ofspecific antibod-ies against human immunodeficiency virus (HIV)is

impor-tantforthedevelopmentofavaccine.One of thepotentially important mechanisms ofprotection against viral spread is

antibody-dependent cellular cytotoxicity (ADCC) (12). In this reaction, HIV-specific antibodiesbind toHIV antigens onthesurface of infectedcells,whicharethenkilled byFc

receptor-positive effector cells. It has been shown that

ADCC to HIV-infected cells is mediated by human immu-noglobulin Gl (IgGl) (13) and also that HIV envelope glycoproteinconstitutes a target for this reaction (12, 15).

HIV type 1 (HIV-1)-specific neutralizing antibodies that areable to inhibit viralinfection invitrohavebeen reported

(24, 28), although the protective role of these antibodies

against HIV infection in humans is controversial. Several authors have reported that HIV-1-neutralizing antibodies canbe producedagainst various regions ofgpl20(7, 11, 16, 19, 21), gp4l (9), and p17 (20). One major site inducing neutralizing antibodies has been described as a

hypervari-able

loop ofgpl20(4,25). Even though several neutralizing

monoclonalantibodies (MAbs) (16, 26) have been produced, noMAbmediating ADCC was previously described.

In this report, we present an MAb which mediates both ADCC and neutralization. The region of

gpl20

towhich it is

directed wasidentified by peptide mapping.

The gpl20 used as an immunogen was prepared from

culturefluidof HIV-1 (human T-cell lymphotropic virus type

III)-infectedH9 cells and was a kind gift from Larry Arthur,

Frederick

Cancer Research Center, Frederick, Md. NMRI mice(NationalVeterinary Institute, Uppsala, Sweden) were

immunizedwithgpl20five times, given at monthly intervals.

Fusion of spleen cells was performed with

Sp20x

Agl4 mouse myeloma cells. MAbs were characterized by iso-types,Westernblot (immunoblot) (27), and immunofluores-cence.

The ADCC was determined as described previously (12, 14). Cells of the monocytoid line U-937 clone 2, chronically

infectedwith HIV-1 strain human T-cell lymphotropic virus typeIIIBused as targets, and peripheral blood mononuclear cells from normal healthy donors as effector cells were

incubatedwith serum or MAb dilutions in a 3-h

51Cr

release assay. The spontaneous release never exceeded 10%. HIV

antibody-positive sera with known ADCC titers as well as

*Correspondingauthor.

seronegative controls including nonimmunized mice were included in each test.

Virus neutralization was performed as follows. Virus supernatant (reverse transcriptase titer, 40.000) was

prein-cubated with serialdilutions (six steps starting with 1:20) of the MAbs for 60min at 37°C. The serum-virus mixturewas added to 5 x

104

peripheral blood mononuclear cells or HUT-78 cells for 60 min at 37°C. After being washed, the cellswerecultured in 96-wellplates for 8 days. Supernatants were then analyzed by HIV antigen capture enzyme-linked immunosorbent assay(ELISA) (V. A. Sundqvist, J. Albert, E. Ohlsson, J. Hinkula, E. M. Fenyo, and B. Wahren, J. Med. Virol., in press). Neutralization wasdefinedas>80% reduction of p24 viral antigen production.

Solid-phase-synthesized (8) 15-amino-acid (aa) peptides with an overlapping sequence of 10 aa representing the complete region of theenvelope (Env) protein based onthe human T-cell lymphotropic virus type "'B sequence (23) wereused asantigens in ELISA. They were a kind gift from J. Rosen (Johnson & Johnson Biotechnology, La Jolla, Calif.). The recombinant proteins pE3(gpl20), pBl (gpl20),

and penv9 (gp4l) were generous gifts from J. Ghrayeb (Centocor, Malvern, Pa.), Scott Putney (Repligen, Corp.,

Cambridge, Mass.), and S. Petteway (Du Pont Co., Rock-ville, Md.),respectively. Amino acid numbering of the Los Alamos database (17) was used.

Peptide ELISA has beendescribedpreviously(3, 27a). To further evaluate thespecificity ofthereactivity, we used the

peptidesassolubleantigenstoinhibitthe MAbreactivityin the ELISA (J. Hinkula, J. Rosen, V.-A. Sundqvist, T.

Stigbrand,and B. Wahren, Mol. Immunol., in press). MAbs werepreincubated with each peptide for 120 min at 37°C and then transferred to pBl-coatedmicroplates, where ELISA wasperformed.

One of the MAbs, P4/D10, was able to mediate high

HIV-specificADCC(Fig. 1). This ADCC activity could also be shown with human HIV antibody-positive sera but not with the other MAbs F58/H3 and T1.1 or with human

HIV-seronegative sera. Nor was the reactivity seen when

P4/D10was testedagainst uninfected target cells.

Of the five MAbs with similarepitopicreactivity and the

abilitytomediate virus neutralization, two are presented in Table 1. In addition, an MAb with a different epitopic

reactivity to the N-terminal end ofgpl20 is presented as a control. The neutralizing capacities ofP4/D10 and F58/H3 936

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-w

0

40~

u,

V-4

ej

30~

20

7

10 *

-30

90 270

810 2430

7290

Ly

Reciprocal

serum

dilutions

FIG. 1. HIV-specificADCC titers forthe MAbsP4/D1OandF58/H3 compared with positive and negative human serum controls. Symbols: [D, P4/D1O;*, F58/H3;

*,

positivehuman sera;

*,

negative human sera; *, Ly.

werehigh (Table 1). Both neutralized human T-cell

lympho-tropic virustype IIIB infectionof peripheral blood lympho-cytes and theCD4+ celllineHUT-78. MAbF58/H3, which wasunable to mediate ADCC, had a high neutralizing titer, while the ADCC-positive P4/D1O MAb had a less efficient

but still significant neutralizing capacity. Neither of the MAbs showed any toxic effects in the assays.

InELISA, P4/D1OandF58/H3showed a strong reactivity withpBl. Fine mapping of theepitopes of these two MAbs was performed with synthetic peptides representing the

wholepBl sequence(Fig. 2a and b). Aspecific reactivity of bothP4/D1OandF58/H3wasfound onlytopeptidesC53 and

C54, which representaa 304to323.

Inaddition, specificpeptideblockingwasperformed (Fig.

2candd).Acomplete inhibition of reactivity of bothMAbs was seen withpeptide C53, while C54 only partlyinhibited

the MAb bindingto pBl.

Thus, an MAb that mediates HIV-specific ADCC and

neutralizationwas identified,and the specificity appearedto be directed toward an epitope located on two

overlapping

peptides, of which the common sequence is IQRGPGRAF (Table 1).GPGRA isaconservedsequencewithinavariable

putative loop region which has been found toinduce neu-tralizing antibodies in animals(25). No clearcorrelationhas

previously been demonstrated between neutralization and ADCC, but it is reasonableto suggestthat thereareseveral ADCC and neutralizing epitopes. However, only one ADCC-mediating epitope at the very C-terminal part of

gpl20has been suggested previously(2). Acorrelationwas

found betweenhigh ADCCtiters and reactivitytoaa304 to 313in a few human sera(unpublished data).

Theimportance oftheisotype ofmurine MAbsmediating ADCC withhuman effector cells varies in different studies.

IgG2a and IgG3 were the predominant isotypes for ADCC (1, 6, 10), butefficient ADCCmediated by IgGl andIgG2b

hasalso been reported (5, 18, 22). ThetwoMAbsdescribed

here reacted with the same peptides but differed in ADCC andneutralizingactivity.Theregion whichinduces neutral-izingantibodies (aa 296 to 331)mightincludealarge propor-tion of the protein, while the epitope inducing

ADCC-mediatingantibodies appearstobealimited sequence within this neutralization-inducing region. -Neutralizing antibodies arebythemselvescapable of

inhibiting

infection

by

binding

directlytothevirus, whereas the

ADCC-mediating

antibod-ies actby bindingtoinfected cells and dependon asecond

binding ofan effector cell.

The

ADCC

activity

might

there-fore bemoresensitivetosmall stericchanges ofthe

binding

site. Other

possible

explanations

for thedifferencein

func-tional activities could be individual variations in the Fc receptorsof the human effector cellsorin the Fc

portions

of the MAbs. Further studies are necessary to characterize

such differences.

Epitope mapping with

sequentially

overlapping

pentade-capeptidesallowsdetermination of

epitopes

of 5to15aabut small variations in the reactivesitenotdetected

by

thelinear

peptides could

apparently

elicit a variation of functional

activities of the

corresponding

antibodies. The

ability

to

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938 NOTES

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FIG. 2. (a and b) Reactivity of the MAbsP4/D1O and F58/H3 diluted1:105tothepeptides described in Table 1. (c and d) Inhibiting effect ofreactivity bypreincubation with the peptides.

selectforfunctionalactivities byselecting regions of interest isimportant for thedevelopment ofa vaccine against HIV. Wegratefully acknowledge Jonathan Rosen, Johnson & Johnson Biotechnology (La Jolla, Calif.), for all the 15-mer peptides and

HansWigzellandCarl HaraldJansonforvaluablediscussions.

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(U U

5-o 0

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1*

44

0,8

c

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U,

,0

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J.VIROL.