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Characterization of a discontinuous human immunodeficiency virus type 1 gp120 epitope recognized by a broadly reactive neutralizing human monoclonal antibody.

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0022-538X/91/116188-06$02.00/0

Copyright C) 1991, American Society forMicrobiology

Characterization of

a

Discontinuous Human

Immunodeficiency

Virus

Type 1

gpl20

Epitope Recognized by

a

Broadly Reactive

Neutralizing Human Monoclonal Antibody

MARKUS THALI,1 UDY

OLSHEVSKY,l

CRAIG

FURMAN,'

DANA

GABUZDA,1

MARSHALL POSNER,2t AND JOSEPH SODROSKI1*

Department ofPathology, Division ofHumanRetrovirology, Dana-Farber CancerInstitute, Harvard Medical School,

Boston,

Massachusetts

02115,1

andBrown

University

MedicalSchool, Providence, Rhode Island 029082 Received 14 May1991/Accepted 29July 1991

While one hypervariable, linear neutralizing determinant on the human immunodeficiency virus type 1 (HIV-1) gpl20 envelope glycoprotein has been well characterized, little is known about the conserved, discontinuous gpl2Oepitopes recognized byneutralizing antibodies in infected individuals. Here, theepitope recognized by a broadly reactive neutralizing monoclonal antibody (F105) derived from an HIV-1-infected patient wascharacterized by examining the effects ofchanges in conserved gpl20 amino acids on antibody reactivity. TheF105epitopewasdisrupted by changesingp120 amino acids 256 and 257, 368 to 370, 421, and 470 to484, whichisconsistentwith thediscontinuous natureof theepitope. Three of theseregionsareproximal tothose previously shown to beimportant for CD4 binding, which is consistent withthe ability of theF105 antibody to blockgpl20-CD4 interaction. Since F105 recognitionwas moresensitive to aminoacidchangesin each of the four identified gpl20 regions than was envelope glycoprotein function, replication-competent mutant viruses that escaped neutralization by the F105 antibody were identified. These studies identify a conserved, functional HIV-1 gpl20 epitope that is immunogenic in man and may serve as a target for therapeutic orprophylactic intervention.

Human immunodeficiency virus (HIV-1) is the etiologic agentof AIDS (1, 7). HIV-1 establishesapersistentinfection in human hosts, eventually resulting in defective cellular

immunitysecondary to CD4 lymphocyte depletion(6). The HIV-1exterior envelopeglycoprotein, gpl20,and the transmembrane envelopeglycoprotein, gp4l, are derived by

cleavage of the gpl60 envelope glycoprotein precursor(6). HIV-1 is tropic for CD4-positive cells by virtue of a high-affinity interaction between the gpl20 exterior envelope

glycoprotein and the CD4 glycoprotein, which acts as the virus receptor (4, 12). Following gpl2O-CD4 binding, the

fusionof viral and host cell membranes, which involves both

gpl20 andgp41 envelope glycoproteins, allows virus entry

(30).

The chronicity of HIV-1 infection implies that the host antiviral immune response is not sufficient to suppress virus

replicationindefinitely. Recent studies of HIV-1 suggest that neutralizing antibodies are an important component of the

protective immune response (2, 5). Two classes of

neutral-izing antibodies are elicited against HIV-1 in infected hu-mans: type restricted and broadly reactive. Type-restricted neutralizing antibodies arise early in infected humans and can bereadily generated in animals by immunization with a variety of preparations ofgpl20 polypeptides (16, 24). The best characterized of the type-restricted neutralizing anti-bodies are those directed against the V3 variable region of

gpl20 (16, 24). Envelope glycoprotein variation within the linear epitope and outside the epitope can allow escape of viruses from neutralization by these antibodies (18, 19). These antibodies do not block CD4 binding but apparently

interferewith post-receptor binding events involved in virus

*Correspondingauthor.

tPresentaddress: Department ofMedicine, New England

Dea-conessHospital, Harvard Medical School, Boston, MA 02115.

entryandsyncytium formation,presumablyacomponentof themembrane fusion process (14, 29).

Later in the course of HIV-1 infection of humans, anti-bodies capable of neutralizing a wider range of HIV-1 isolates appear (26, 32). Thesebroadly neutralizing antibod-ies havebeendifficulttoelicitin animals andare notmerely

theresultof additive anti-V3loopreactivitiesagainstdiverse HIV-1isolates that accumulateduring active infection (23).

A subsetof the broadly reactive antibodies, found inmost HIV-1-infected individuals, interferes with the binding of gpl20and CD4 (17, 28). This activity isobservedonlyatlow

dilutions of patient sera, suggesting that the titer and/or affinity of these antibodies is low. These antibodies are present in individuals whose serumreacts only with native

gpl20,notwith reducedgpl20, suggestingthatatleastsome of these antibodies recognizediscontinuous gpl20 epitopes (8).Thediscontinuousnatureoftheepitopesand the mixture

of different antibodies found in patient serum have made

characterization ofthe epitopes recognizedbybroadly

neu-tralizing antibodies difficult. Recently, human monoclonal antibodiesderived fromHIV-1-infected individualsthat

rec-ognize the gpl20 glycoproteins from a diverse range of HIV-1 isolates, that block gpl20-CD4 binding, and that neutralize virus infection have been identified (11, 22, 27). Here we characterize the epitope recognized by one such human monoclonal antibody, designated F105 (22). The F105antibodyrecognizesthe

gpl20

glycoprotein of theIIIB,

MN, RF, CC, and ARV-2 strains of HIV-1, blocks

gpl20-CD4binding,and hasbeen showntoneutralize the IIIB and MN HIV-1isolates (21a, 22).

MATERIALSANDMETHODS

Immunoprecipitation ofHIV-1 gpl20 mutants. Mutations were introduced into the HXBc2 env gene and mutant

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glycoproteins were transiently expressed in COS-1 cells as

previously described (20). Cell lysates radiolabelled with

[35S]cysteinewereprecipitatedeither with the F105antibody

or with pooled serum derived from patients with AIDS.

Immunoprecipitates ofwild-type and mutant glycoproteins

were analyzed on sodium dodecyl sulfate-polyacrylamide

gels and the relative intensity of the envelope glycoprotein bands assessed bydensitometric scanningof the

autoradio-graphs as previously described (20). The F105 recognition index for a given mutant glycoprotein was calculated by

usingthe following formula:

F105 recognitionindex =

mutant

(gpl60

+

gpl20)

wild-type (gpl60 +

gpl20)F

(wild-type (gpl60

+

gpl20)\

x patientserum

mutant(gpl60 +

gpl2O)/

Neutralization ofmutant virusesby the F105 antibody. To

quantitate the ability of the F105 antibody to neutralize

HIV-1virusesincorporatingmutantenvelopeglycoproteins,

an envelope glycoprotein complementation assay was

em-ployed(9). Briefly,COS-1 cells werecotransfectedwith the

plasmid expressing wild-type or mutant envelope glycopro-teins and a plasmid containing an env-defective HIV-1

provirus encodingthebacterialchloramphenicol acetyltrans-feraseCATgene (9). COS-1 cellsupernatants containingan

equivalent numberofreverse transcriptase units of recom-binant virions for each mutant were divided into two

frac-tions. Each fraction was incubated at 37°C for 1 h in the

presence or absence ofa high concentration (80,ug/ml) of

purified F105 antibodyprior toincubation withJurkat

lym-phocytes.Threedays after infection, Jurkat cellswerelysed

andCATactivity wasmeasured.

RESULTS

Mapping

gpl20

residues important for F105 recognition. Since the F105antibody recognizes divergentHIV-1isolates (22), amino acids conserved among HIV-1 strains must

constitutethecriticalcomponentsofthediscontinuousF105

epitope. Toidentify

gpl20

amino acids important for

recog-nition by the

F105

antibody, the reactivity ofthe antibody with a set of HIV-1

gpl20

mutants altered in conserved residues was examined. These mutant glycoproteins have

beenpreviously characterized withrespect to rate of

gpl60

precursorprocessing,

subunit

association, andCD4-binding ability, allowing an assessment ofthe conformational cor-rectness of the mutantproteins (10, 20). Radiolabelled cell lysates from COS-1 cells transfectedwith

plasmids

express-ing the wild-type or mutant envelope

glycoproteins

ofthe

HXBc2 strain ofHIV-1 were

precipitated

either with F105 antibody or with a mixture of sera derived from HIV-1-infected humans. Since the mixed patient sera

recognize

multiple

gp120

epitopes, mostof which are notaffected

by

the amino acid changes in the mutant

glycoproteins,

the latterprecipitation allows an assessment ofthe amount of

mutantenvelope

glycoproteins

present inthe cell

lysate.

The F105 recognition index, which represents the

ability

ofa

given

mutant envelope

glycoprotein

tobe

recognized by

the F105 antibody relative to that of the

wild-type

envelope

glycoprotein,

wascalculated as described in Materials and Methods.

Theresults ofthe

immunoprecipitation

studiesareshown

TABLE 1. F105recognition indicesandrelativeCD4-binding abilities of selectedHIV-1gpl20mutantsa

Mutant

Wild type... 102E/L... 113D/R... 117K/W... A119205 ... 120/121VKILE... 125L/G... 252R/W... 256S/Y... 257T/R... 257T/A... 257T/G... 262 N/T... 266A/E... 267 E/L... 269 E/L... 356N/I... 368D/E... 368D/T... 368D/P... 368D/R... 368D/N... 368D/K... 370E/D... 370E/Q... 370E/R... 380/381 GE/YW... 382F/L... 384YIE...

386N/Q... 395W/S... 420I/R... 421 K/L... 427W/S... 427W/V... 456R/K... 457D/A... 457D/R... 457D/E... 457D/G... 463N/D... 465S/L ... 470P/G... 475M/S... 477D/V... 482/482/484 ELY/GRA... 485 K/V... 491 I/F...

F105recognitionindexb

(RelativeCD4-bindingabilityc) 1.00(1.00) 0.45 0.92 0.60 >1.5 (1.4) 1.21e 0.67 >1.5 <0.025 (0.30) <0.0072 (0.16) <0.078 (1.12) <0.025 (1.04) 0.60 >1.5 0.80 0.76 >1.5 <0.024(0.09) <0.015(0.33) <0.015(0.09) <0.013(<0.004) 0.079(0.19) <0.02(<0.005) <0.017 (0.45) <0.038(0.018) <0.0075 (<0.003) 1.5 0.54 0.159 1.0.0 0.44 >1.5 <0.020(0.55) >1.5(<0.006) >1.5(<0.012) >1.5 0.93 (0.09) 0.42(0.15) 1.5 0.89 1.1 >1.5 0.19(0.82) <0.013(1.03) 0.15 (0.39) 0.018(0.44) >1.5 0.64

a Other

gpl20

mutantstested forF105recognitionincluded 40Y/D,69W/L,

76P/Y, 80N/R,88 N/P,103 Q/F,106 E/A, 113 D/A, 207 K/W,298R/G,

308/309/310RIQ/RPELIPVQ,314G/W,314G/Q,380G/F,381E/P,386N/R,

392N/E+397N/E,406N/G,429K/L,430V/S,432K/A,433A/L,435Y/H,

435Y/S,438 PIR,450T/N,493 P/K,495G/K,497/498/499 APT/VLL,and

500/501 KA/KGIPKA. Precipitation of eachof these mutantsby the F105

antibody wasatleastasefficientasthatseenfor thewild-typeglycoproteins.

bEachvaluefor therecognitionindexrepresents themeanofatleasttwo

independentexperiments,withexperimentalvariationtypicallynot morethan

15% ofthevaluereported.

RelativeCD4-bindingabilities ofmutantglycoproteinswere taken from

reference20.

d The A119-205mutantcontainsadeletion oftheentireV1-V2regionsof

HIV-1gpl20.Thepredictedaminoacidsequence and the residue numbernear

thedeletion isLeu-116Lys-117-Pro-118 Gly-Pro-206Lys-207-Val-208

Ser-209.

e Theimmunoprecipitationof thegpl20form of thismutantglycoprotein by

the F105 antibody was decreased relative to that ofthe wild-type gpl20 glycoproteins, althoughprecipitation of thegpl60form of themutantwas

slightlymoreefficientthanthatof thewild-typeglycoprotein. (

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J . Lnt

100

EZIZIFI1

iA

1..

I

200

C2

nil

LO

I

r2ri" [s- m- sLl El XILF, "

300 400 500

C3

fC4

10C5

0.2.2

0.6

0)

cc1.4

LO

LL 1.8 0)

~2.2

FIG. 1. Effects of amino acidchanges in HIV-1 gp120onrelativeCD4-binding ability and theF105recognitionindex. The linearsequence

ofthe HIV-1gp120 molecule is shown, withthe conserved regions in light andthe variable regions in dark shading.The positions of the signal

sequence(S) andthe conserved regions (CltoC5)areindicated, as aretheamino acidnumbers. Amino acid numbering is basedonresidue

1correspondingtotheinitial methionine.Above thegpl20linearmapis plottedthenegativelogof the relative CD4-binding ability observed

for themostdisruptive changeatagiven aminoacid. Valuesarederivedfrom reference20. Theopenbarsindicatemutantglycoproteins that exhibitedindices forgpl60precursorprocessingorgpl20-gp4lassociation less than 40% of those of thewild-type values. Beneath thegp120 linearmapis plottedthenegativelog of the recognition indexfor the FlOS antibody observed for themostdisruptive changeatagivenamino

acid.

in Table 1 and Fig. 1. The F105 antibody precipitated both thegp160 and the gp120 forms of the majority of themutants at least as well as it did the wild-type envelope

glycopro-teins. Mutantglycoproteins with changes in amino acids256 and 257,368to370, 421, or470to 484exhibited significant reductions in the ability to be precipitated by the FlOS antibody. Incasesinwhichmultipleaminoacidsubstitutions

atasinglegpl20 residuewereexamined, all of the changes in

the above four regions resulted in significant decreases in F105 recognition. The global conformation of most of the gp120 mutants exhibiting decreased F105 recognition was

not grossly altered, as judged by the rate of envelope

precursorprocessing, gpl20-gp4l association, CD4 binding, orfunctional studies (see below) (10, 20).

Neutralization of variant viruses bytheF105antibody. For severalof thegp120 changes, CD4 bindingwasnotaffected to the same degree as was F105 recognition. This, in

conjunction with the observation that substantial decreases in CD4 binding ability can be tolerated by

replication-competentviruses (31), suggested the possibility that some

ofthese mutants might escape neutralization by the FlOS

antibody. To test this, we employed an assay in which an

env-defective HIV-1 provirus encoding the bacterial CAT

genewascomplementedforasingleround of replication by

the wild-type or mutant envelope glycoproteins (9). The F105antibodywasaddedtotherecombinant virions priorto incubation of the virus-antibody mixture with targetJurkat lymphocytes. Viruses containing the wild-type envelope

glycoproteins were neutralized by the FlOS antibody, as were virusescontainingmutantenvelopeglycoproteins that

were recognized as well as the wild-type glycoproteins by

the F105 antibody (Fig. 2). By contrast, viruses containing

the257T/G, 257T/R, 368D/N, 368D/1, 370E/D,370E/Q,

421 K/L,and 475 M/Smutant envelopeglycoproteinswere

significantly more resistant to neutralization by the F105 antibody compared with virions containing the wild-type

glycoproteins. The 470 P/G and 477 D/V mutant glycopro-teins, which exhibited F105 recognition indices between those ofthewild-type and475 M/Sglycoproteins,exhibited

an intermediate level ofsensitivity to F105 neutralization. No obvious relationship between the ability of a given

mutantglycoproteintobeneutralized bythe FlOS antibody 2

1

0) c

._

c co

m

0 U1)

._

-i I)

-J

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100 _ 89 102 100

95

90 ~~~89

INA

.'Sy8

82

77

70 t 12/2 25i56 9 6 6 7 7 8 2 5 5 7 7 7

<60

o ~~~~~~~~~~~~~~~~~50

50

* 40

.~30

24 21

20

10 7.2 7.2 6.7

4.9 4.1 3.4 3.8

w.t. 120/121 257 257 266 298 368 368 370 370 386 421 457 457 470 475 477 VK/LE T/G T/R A/E R/G D/N D/T E/D E/Q N/Q K/L D/A D/G P/G M/S DN

FIG. 2. Resistance to F105 neutralization for some HIV-1 gpl20 mutants. Recombinant viruses containing the mutant envelope glycoproteins and packaging the enm-defective provirus encodingthe bacterial CAT gene were produced in COS-1 cells as described in Materials and Methods. The virions were incubated in the presence or absence of purified F105 antibody prior to incubation with Jurkat lymphocytes. Thepercentage of the CAT activity observed in the Jurkat target cells for each mutant following incubation with F105 antibody relative to the CAT activity observed in the absence of antibody is shown.Avalue of 0 indicates complete neutralization, while a value of 100indicates no neutralization. The experiment was repeated twice with similar results. The uninhibited abilities of the mutant envelope glycoproteins to complement virus entry into Jurkatlymphocytesrelative to a value of 100 for the wild-type envelope glycoproteins were as follows: 120/121VK/LE, 86; 257 T/G,36;257T/R,40; 266A/E,72;298R/G,77;368D/N, 14; 368D/T,32; 370E/D,87;370E/Q,33;386N/Q, 100; 421 K/L, 29; 457 D/A, 36; 457D/G, 57;470P/G,69; 475M/S, 97;and 477 D/V,69 (31). w.t., wild type.

and the relative ability ofthe mutant glycoprotein to

com-plement virus entry into Jurkat lymphocytes was observed

(Fig. 2). Some of the F105-resistant mutants (257 T/G and 475 M/S) weretestedfor sensitivity to neutralization by the 0.5r monoclonal antibody, which recognizes theV3loopof HIV-1gpl20 (15). Thesemutantsexhibited neutralization by the 0.53 antibody comparable to that of the wild-type envelope glycoproteins (datanotshown), indicating that the

escapefrom F105 neutralization was antibody specific.

DISCUSSION

Amino acid changes in HIV-1 gpl20 residues located in fourdiscontinuousregionsresulted indramaticreductionsin recognition byabroadly reactive neutralizinghuman

mono-clonal antibody, F105. That multiple substitutions in the

same residues reduced F105 recognition in the apparent absence ofglobal conformational disruption ofgpl20 and thatfunctionalneutralizationescapemutantsweregenerated

bysomechangesineach of theseregionssupportamodel in

which these four regionsconstitute criticalelementsnear or

within theF105 epitope. This model is consistent with that of other characterized discontinuous epitopes on proteins,

which are typically composed of 13 to 24 amino acids derived from two tofive continuous components (13).

Significant overlap between gpl20 regions implicated in CD4 binding and those important for F105 recognition exists, which is consistent with the ability of the F105 antibodytoblockgpl20-CD4interaction(22). Twoelements importantfor F105 recognition atpositions 256 and257 and 368to 370correspondprecisely togpl20aminoacids previ-ouslyidentifiedasimportantforbindingCD4(20). While all of the different amino acid substitutions in these residues significantly disrupted F105 recognition, a number of the

substitutions exerted only small effectsonCD4binding.The third element importantfor F105recognitionatlysine421 is adjacent in the linear sequence to tryptophan 427, changes in which results in dramatic reductions in CD4-binding ability (3, 20)but not F105recognition. Tryptophan 427resides ina more hydrophobic region than does lysine 421, suggesting

that CD4 interacts with a less accessible segment of the fourth conserved region ofgp120 than does the F105 anti-body. The inclusion of these three discontinuous regions, previouslyimplicated inCD4binding(20), withinor near an

antibody epitope suggests that they are proximal on the nativeglycoprotein.

The fourth element important for F105 recognition at

gpl20 residues 470to 484 does notoverlap the region near

aspartic acid 457 implicated in CD4 binding (20). Both

hydrophilic regions, which symmetrically flank the short fifth variableregionofgpl20,exhibit strong 3-turn

potential,

which could result in the apposition of theseregions in the nativeglycoprotein. Further work is required to

verify

this

possibility.

A mutant

(M119-205)

containinga largedeletion

spanning

thecarboxylportionofCl and theV1-V2variableregionsof

gpl20 retained CD4-binding ability and F105

recognition.

This result demonstrates that thisregion,which includes the

peptideT region(21) ofgpl20(residues 190to 197), is not a necessary component of either the CD4-binding

moiety

or the F105 epitope. Changes in the other variable

regions

of

gpl20, although not as

comprehensive

as the

zv119-205

deletion, did not decrease F105

recognition,

which is

con-sistent with the ability of the

antibody

to

recognize

diverse HIV-1 isolates(22).

Although other mechanisms have notbeen ruled out, the available data suggestthat interference with CD4

binding

is a major mechanism of in vitro virus neutralization

by

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F105antibody.Since

gpl20

structuralrequirementsforF105 binding and virus replication were notidentical, neutraliza-tion escape byvariation within the

gpl20

regions important for F105 recognition was possible. The high degree of

conservation of these regions observed in HIV-1 isolates

suggeststhatconstraintsonsuchchangeexist;thatselective

pressureforchangein theseregionsislow,perhapsbecause oflowantibody titer;orthat other mechanisms for

neutral-ization escape exist. It has been observed that HIV-1

mu-tants resistantto neutralization by infected-patientseracan

be selected in tissue culture, apparently as aresult ofvirus

variationoutside of the epitope (25). Further studies willbe

required to assessthe potentialof HIV-1 for in vivo escape

from neutralization by antibodies such as F105 that

recog-nizeconserved,functional epitopes.

This initial characterization of an HIV-1

gpl20

epitope

recognized by a broadly reactive human neutralizing

anti-body may provide a reference point for analysis of other suchantibodies andcouldaid in theexplorationofmeansto

efficientlyelicit thistype ofantibody response.

ACKNOWLEDGMENTS

We thankRobert Gallo, FlossieWong-Staal, Max Essex, Bruce

Walker, Shuzo Matsushita, and T. Hattori for reagents, Ginny

Nixonformanuscript preparation, andAmyEmmert for artwork. Markus Thali was supported by the Swiss National Science

Foundation. UdyOlshevsky performedthis work whileon

sabbat-ical from the Israel Institute for Biological Research, Nes-Ziona,

Israel. Dana Gabuzda was supported by an AIDS Postdoctoral

Training Award from the National Institutes of Health. Marshall

Posner was supported by the National Institutes of Health (grant

A126926). JosephSodroskiwassupported bythe LeukemiaSociety

of America, the Aaron Diamond Foundation, and the National

Institutes of Health(grantsA124755 andA131783).

ADDENDUM

Another human monoclonal antibody (1125 H), derived

from an HIV-1-infected individual, that blocks gpl20-CD4

bindingandneutralizesanumberofdivergentHIV-1isolates

has recently been described(31a).

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Figure

TABLE 1. F105 recognition indices and relative CD4-bindingabilities of selected HIV-1 gpl20 mutantsa
FIG.1.forofexhibitedsequencelinear1 corresponding the Effects of amino acid changes in HIV-1 gp120 on relative CD4-binding ability and the F105 recognition index
FIG. 2.glycoproteinsfollows:glycoproteinsMaterialsrelativelymphocytes.100;100 Resistance to F105 neutralization for some HIV-1 gpl20 mutants

References

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