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
CRAIGFURMAN,'
DANAGABUZDA,1
MARSHALL POSNER,2t AND JOSEPH SODROSKI1*Department ofPathology, Division ofHumanRetrovirology, Dana-Farber CancerInstitute, Harvard Medical School,
Boston,
Massachusetts02115,1
andBrownUniversity
MedicalSchool, Providence, Rhode Island 029082 Received 14 May1991/Accepted 29July 1991While 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
6188
on November 10, 2019 by guest
http://jvi.asm.org/
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 mustconstitutethecriticalcomponentsofthediscontinuousF105
epitope. Toidentify
gpl20
amino acids important forrecog-nition by the
F105
antibody, the reactivity ofthe antibody with a set of HIV-1gpl20
mutants altered in conserved residues was examined. These mutant glycoproteins havebeenpreviously 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 transfectedwithplasmids
express-ing the wild-type or mutant envelope
glycoproteins
oftheHXBc2 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 serarecognize
multiple
gp120
epitopes, mostof which are notaffectedby
the amino acid changes in the mutant
glycoproteins,
the latterprecipitation allows an assessment ofthe amount ofmutantenvelope
glycoproteins
present inthe celllysate.
The F105 recognition index, which represents theability
ofagiven
mutant envelopeglycoprotein
toberecognized by
the F105 antibody relative to that of thewild-type
envelope
glycoprotein,
wascalculated as described in Materials and Methods.Theresults ofthe
immunoprecipitation
studiesareshownTABLE 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. (
on November 10, 2019 by guest
http://jvi.asm.org/
[image:2.612.314.554.110.566.2]J . Lnt
100
EZIZIFI1
iA
1..
I
200
C2
nil
LOI
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
on November 10, 2019 by guest
http://jvi.asm.org/
[image:3.612.72.566.71.419.2]100 _ 89 102 100
95
90 ~~~89
INA
.'Sy8
8277
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
thispossibility.
A mutant
(M119-205)
containinga largedeletionspanning
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 variableregions
ofgpl20, although not as
comprehensive
as thezv119-205
deletion, did not decrease F105
recognition,
which iscon-sistent with the ability of the
antibody
torecognize
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 neutralizationby
theon November 10, 2019 by guest
http://jvi.asm.org/
[image:4.612.146.484.70.284.2]F105antibody.Since
gpl20
structuralrequirementsforF105 binding and virus replication were notidentical, neutraliza-tion escape byvariation within thegpl20
regions important for F105 recognition was possible. The high degree ofconservation 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
epitoperecognized 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).
REFERENCES
1. Barre-Sinoussi,F., J.C. Chermann,F.Rey,M. T.Nugeyre,S.
Chamaret,J.Gruest,C.Dauget,C.Axler-Bin,F.Vezinet-Brun,
C. Rouzioux, W.Rozenbaum,and L. Montagnier. 1983.
Isola-tion of a T-lymphocyte retrovirus from a patient at risk for
acquired immunodeficiency syndrome (AIDS). Science 220:
868-871.
2. Berman, P.,T.Gregory,L.Riddle,G.Nakamura,M.Champe,
J.Porter,F.Wurm,R.Hershberg,E. K.Cobb,andJ.Eichberg.
1990.Protection ofchimpanzeesfrominfectionbyHIV-1 after vaccinationwithrecombinantglycoproteingpl20butnotgpl60.
Nature(London)345:622-625.
3. Cordonnier, A.,L. Montagnier,and M.Emerman. 1989. Single
amino acid changes inHIV envelope affect viral tropismand receptorbinding.Nature (London)340:571-574.
4. Dalgleish, A. G., P. C. L. Beverly, P. R. Clapham, D. H.
Crawford, M. F.Greaves,andR. A.Weiss. 1984.TheCD4(T4)
antigenis anessentialcomponentofthereceptor for theAIDS
retrovirus. Nature(London)312:763-767.
5. Emini, E.,P.Nara, W.Schlief, J. Lewis, J. Davide,D.Lee, J.
Kessler,S.Conley,M. Matsushita,S.Putney,R.Gerety, and J.
Eichberg. 1990. Antibody-mediated in vitro neutralization of
humanimmunodeficiency virus type 1 abolishes infectivity for
chimpanzees.J. Virol. 64:3674-3678.
6. Fauci, A., A. Macher, D. Longo, H. C. Lane, A. Rook, H.
Masur, and E. Gelmann. 1984. Acquired immunodeficiency
syndrome:epidemiologic, clinical, immunologic,and
therapeu-tic considerations. Annu. Int.Med. 100:92-106.
7. Gallo,R.C.,S. Z.Salahuddin,M. Popovic,G. M.Shearer, M. Kaplan,B. F. Haynes, T.J. Palker, R. Redfield, J. Oleske, B. Safai,G.White,P.Foster,and P. D. Markham. 1984.Frequent detection and isolation ofcytopathic retroviruses (HTLV-III) frompatientswith AIDS and at risk for AIDS. Science 224:500-503.
8. Haigwood,N., C.Barker, K.Higgins,P. Skiles, G.Moore, K. Mann, D.Lee,J. Eichberg,and K. Steimer. 1990. Evidence for neutralizingantibodies directedagainstconformationalepitopes of HIV-1gp120. Vaccines 90:313-320.
9. Helseth,E.,M.Kowalski,D.Gabuzda,U.Olshevsky,W. Hasel-tine, and J. Sodroski. 1990. Rapid complementation assays measuringreplicativepotential of HIV-1envelopeglycoprotein
mutants.J. Virol.64:2416-2420.
10. Helseth, E., U. Olshevsky, C. Furman, andJ. Sodroski. 1991. Human immunodeficiency virus type 1 gp120 envelope glyco-protein regions important for association with thegp4l trans-membraneglycoprotein. J. Virol. 65:2119-2123.
11. Ho, D.,J. McKeating,X. Li,T. Moudgil,E.Daar, N.-C. Sun, andJ.Robinson. 1991. Conformationalepitopeongp120
impor-tantin CD4binding and humanimmunodeficiencyvirustype1 neutralization identified by a human monoclonal antibody. J. Virol. 65:489-493.
12. Klatzmann, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T.Hercend,J.C.Gluckman,and L.Montagnier.1984. T-lymphocyte T4 molecule behaves asthe receptor for human retrovirusLAV. Nature (London) 312:767-768.
13. Laver, W. G., G. Air, R. Webster, and S. Smith-Gill. 1990. Epitopes onproteinantigens: misconceptions and realities. Cell 61:533-556.
14. Linsley, P., J. Ledbetter, E. Thomas, and S.-L. Hu. 1988. Effects ofanti-gpl20 monoclonal antibodiesonCD4 receptorbindingby the env protein of human immunodeficiency virus type 1. J. Virol.62:3695-3702.
15. Matsushita, M., M. Robert-Guroff, J. Rusche, A. Koito, T. Hattori, H. Hoshino, K. Javaherian, K. Takatsuki, and S. Putney. 1988. Characterization ofahuman immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope. J. Virol. 62:2107-2144.
16. Matthews, T., A. Langlois, W. Robey, N. Chang, R. Gallo, P. Fischinger, and D. Bolognesi. 1986. Restrictedneutralization of divergent human T-lymphotropic virus type III isolated by antibodies to the major envelope glycoprotein. Proc. Natl. Acad. Sci. USA 83:9709-9713.
17. McDougal,J. S.,J. Nicholson, G. Cross, S. Cort, M. Kennedy, and A. Mawle. 1986. Binding of the human retrovirus HTLV-III/LAV/ARV/HIV to the CD4 (T4) molecule: conformation dependence, epitope mapping, antibody inhibition, and poten-tial foridiotypic mimicry. J. Immunol. 137:2937-2944. 18. McKeating,J., J.Gow, J. Goudsmit, L. Pearl, C. Mulder, and R.
Weiss. 1989. Characterization ofHIV-1 neutralization escape
mutants.AIDS3:777-783.
19. Nara, P., W. Robey, L. Arthur, D. Asher, A. Wolff, C. Gibbs, D. C.Gajdusek,and P.Fischinger. 1987. Persistent infection of chimpanzeeswith human immunodeficiencyvirus: serological responses and properties of reisolated viruses. J. Virol. 61: 3173-3180.
20. Olshevsky, U.,E.Helseth, F. Furman, J. Li, W. Haseltine, and J. Sodroski. 1990. Identification of individual HIV-1gpl20amino acids important for CD4 receptor binding. J. Virol. 64:5701-5707.
21. Pert,C., J. Hill, M. Ruff, R. Berman, W. Robey, L. Arthur, F. Ruscetti,and W.Farrar. 1986. Octapeptides deduced from the neuropeptide receptor-like pattern of antigen T4 in brain po-tently inhibithuman immunodeficiency virus receptor binding and T-cell infectivity. Proc. Natl. Acad. Sci. USA 83:9254-9258.
21a.Posner,M. Unpublisheddata.
22. Posner, M., T. Hideshima, T. Cannon, M. Mukherjee, K. Mayer, andR. Byrn. 1991. An IgG human monoclonal antibody that
reacts with HIV-1 gpl20, inhibits virus binding to cells, and
neutralizesinfection. J. Immunol. 146:4325-4332.
on November 10, 2019 by guest
http://jvi.asm.org/
23. Profy, A., P. Salinas, L. Eckler, N. Dunlop, P. Nara, and S. Putney. 1990. Epitopes recognized by the neutralizing antibod-iesofanHIV-1-infected individual.J.Immunol. 144:4641-4647.
24. Putney, S., T. Matthews, W.G. Robey, D. Lynn, M.
Robert-Guroff, W. Mueller, A. Langlois, J. Ghrayeb, S. Petteway, K. Weinhold, P. Fischinger, F. Wong-Staal, R. Gallo, and D. Bolognesi. 1986. HTLV-III/LAV-neutralizing antibodies to an
E. coli-producedfragment of the virusenvelope. Science234: 1392-1395.
25. Reitz, M., C. Wilson, C. Naugle, R. Gallo, and M. Robert-Guroff. 1988.Generation ofaneutralization-resistant variant of
HIV-1is dueto selection forapoint mutation in the envelope gene. Cell54:57-65.
26. Robert-Guroff, M., M. Brown, and R. Gallo. 1985. HTLV-III neutralizing antibodies in patients with AIDS andAIDS-related complex. Nature(London) 316:72-74.
27. Robinson, J., D. Holton, S. Pacheco-Morell, J. Liu, and H. McMurdo. 1990. Identification of conserved and variant epitopes of HIV-1 gpl20 by human monoclonal antibodies produced by EBV-transformed cell lines. AIDS Res. Hum. Retroviruses 6:567-580.
28. Schnittman, S., H. C. Lane, J. Roth, A. Burrows, T. Folks, J. Kehrl, S.Koenig, P. Berman, and A. Fauci. 1988.
Characteriza-tion ofgp120 bindingtoCD4 andan assaythatmeasuresability ofseratoinhibit thisbinding. J.Immunol. 141:4181-4186. 29. Skinner, M., A. Langlois, C. McDanal, J. S. McDougal, D.
Bolognesi, andT.Matthews. 1988. Characteristicsofa
neutral-izingmonoclonal antibodytotheHIVenvelopeglycoprotein.J. Virol. 62:4195-4200.
30. Stein, B., S. Gouda, J. Lifson, R. Penhallow, K. Bensch, andE. Engleman. 1987. pH-independentHIVentry into CD4-positive Tcells via virus envelope fusiontotheplasma membrane.Cell 49:659-668.
31. Thali, M., U. Olshevsky, C. Furman, D.Gabuzda, J. Li, andJ. Sodroski. 1991. Effects of changesingpl2O-CD4-bindingaffinity
onhumanimmunodeficiency virustype1envelopeglycoprotein function and soluble CD4 sensitivity. J. Virol. 65:5007-5012. 31a.Tilley, S. A., W. J. Honnen,M. Racho, M. Hilgartner,and A.
Pinter. A humanmonoclonal antibody againsttheCD4binding site of HIV-1gpl20 exhibitspotent,broadly neutralizing activ-ity. Res. Virol., inpress.
32. Weiss, R., P. Clapham, R. Cheingsong-Popov,A. Dalgleish, C. Carne, I. Weller, andR.Tedder. 1985. Neutralizationof human T-lymphotropic virus typeIII bysera ofAIDS and AIDS-risk patients. Nature(London) 316:69-72.