AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 6, 1992 Mary Ann Liebert, Inc., Publishers

Identification of Two Neutralizing and 8 Non-Neutralizing Epitopes on Simian Immunodeficiency Virus Envelope Using Monoclonal Antibodies K.A.

KENT,1

E.

RUD,2 T. CORCORAN,1

C. POWELL,1 C. EJ. STOTT1

THIRIART,3 C. COLLIGNON,3,4 and

ABSTRACT Ten new monoclonal antibodies (MAbs) to SIV envelope were produced and characterized. Using a panel of 28 MAbs, 10 antibody binding sites on SIV envelope protein were identified. Seven sites were located in gpl20 and three in gp41. Five sites in gpl20 and two in gp41 were defined by overlapping peptides. The remaining two sites on gpl20 and one on gp41 were distinguished by competition binding assays but could not be defined by overlapping peptides, suggesting that they were discontinuous or conformational epitopes. Five of the 28 MAbs consistently and reliably neutralized the infectivity of SIVmac2S1. Two of these bound to a peptide (aal71-190) in the V2 region. The remaining three MAbs bound to a conformational epitope on gpl20. These two neutralizing epitopes on SIV are analogous to similar epitopes recently described in HIV-1. In contrast, three MAbs binding to the V3 region of SIV failed to neutralize infectivity, suggesting that this region in SIV may by functionally different from the V3 loop in HIV-1.

INTRODUCTION

Recombinant SIV vaccines are also being evaluated. In a study4 vaccination with live recombinant vaccinia virus expressing envelope of SIVmne followed by boosting with partially purified gpl60 derived from recombinant baculovirus protected 4 of 4 macaques challenged with a biological clone of SIVmne. Passive transfer of sera from animals vaccinated or infected with HIV-2 or SIV also protected monkeys against challenge,5 suggesting that protection may be conferred by antibody alone. Experiments with HIV and SIV in animals clearly suggest a role for antibody to envelope in protection. If the SIV model is to be of use in developing candidate HIV envelope vaccines it is essential to define epitopes with biological activity, and thus identify the important similarities and differences between the envelopes of SIV and HIV. One neutralizing epitope on SIV transmembrane protein6 has been described. In an earlier report,7 we described the producrecent

SIMIAN

IMMUNODEFICIENCY virus (SIV) infection of macaques is a valuable tool for studying the pathogenesis of lentivirus infection and for evaluating the efficacy of vaccines. Vaccines composed of human immunodeficiency virus type 1 (HIV-1) envelope as a recombinant protein have successfully protected chimpanzees against challenge.1,2 A recombinant gpl20 protein successfully protected two chimpanzees2 and this protection was correlated with the presence of neutralizing antibody to the third hypervariable domain (V3 loop). However, a recombinant gpl60 protein also containing the V3 loop did not confer protection. Passive transfer of a monoclonal antibody against HIV-1 V3 loop also protected chimpanzees against intravenous challenge.3 Thus, there is evidence that antibody to the V3 loop can protect against challenge with the IIIB strain of HIV-1.

'National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts, England. 2Wellcome Research Laboratories, Langley Court, Beckenham, Kent, England. 3SmithKline Beecham Biologicals s.a., Rue de l'Institut 89, B-1330 Rixensart, Belgium. 4New England Regional Primate Research Centre, Harvard Medical School, One Pine Hill Drive, P.O. Box 9102, Southborough,

01772-9102.

1147

MA

KENT ET AL.

1148 tion and characterization of 18 MAbs to SIVmac25] envelope proteins, 13 MAbs to gpl20, and 5 MAbs to gp32. Of the 18 MAbs 4 had neutralizing activity. We now describe 10 new monoclonal antibodies and, using the panel of 28 MAbs, define 10 B-cell epitopes on SIVmac25, gpl20 and gp32, 2 of which elicit neutralizing activity.

MATERIALS AND METHODS Cell lines and viruses Cell lines were maintained at 1-5 x 105 cells/ml in RPMI1640 supplemented with 8% heat-inactivated fetal calf serum as previously described.7 SIVmac25i and SIVcynl86 were obtained from Dr. R. Desrosiers, New England Regional Primate Centre, Boston, MA; SIVsm.7 was obtained from Dr. P. Fultz, Yerkes Primate Centre, Atlanta, GA; SIVsrnmB670 was obtained from Dr. M. Murphy-Corb, Delta Regional Primate Centre, Covington, LA; and HIV-2 SBL-6669 was obtained from Professor G. Biberfeld, National Bacteriological Laboratory, Stockholm, Sweden. SIVmac251 32H (11/88 pool) was isolated fromarhesus macaque (32H) infected with SIVmac251 and cultured in C8166 cells to provide a low-pass pool of stock virus.8 Virus for neutralization assays was grown routinely as a persistent infec¬ tion in HUT-78 cells whereas virus for all other assays was grown as an acute infection in C8166 cells.7

Preparation and characterization of MAbs Eighteen of the MAbs used in this investigation were those described by Kent et al.7 Ten new MAbs were generated and characterized by ELISA, surface fluorescence, radioimmune precipitation assay, Western blot, and immunoglobulin (Ig) isotype as previously described.7

Neutralization assays Neutralization assays

viously.7

were

performed

as

described pre¬

Peptide mapping The seven peptides shown in Table 1 were predicted to be immunogenic from analysis of sequence data using the method developed by Hopp and Woods16 based on hydrophilicity

Table 1. Location

Peptide No.

Amino acid no.

1003 1004 1005 1006 954 738 1008

170-189 311-343 435-454 517-536 594-616 738-770 855-874

and

Amino Acid

Sequence

profiles. These data were used in conjunction with cysteine alignments of the envelope proteins of HIV-1, HIV-2, and SIV to identify regions of the SIV genome analogous to immuno¬ genic regions of HIV-1 envelope. A series of overlapping 20mers, with an overlap of 10 amino acids, spanning the whole of gpl20 (EVA 774) or gp41 (EVA 798) were obtained from the Reagent Program of the European Vaccine against AIDS (EVA) Project. Three of the 49 overlap¬ ping peptides of EVA774 (5, 38, and 46) were not available when this study was undertaken and these peptides were not tested. The reactivity of MAbs with all the peptides was determined by ELISA. ELISA plates were coated with peptides at a concentration of 2.5 µg/ml in DDW and allowed to dry over¬ night at 37°C. MAbs, as undiluted culture supernatant fluids, were tested. Bound antibody was detected using affinity-puri¬ fied goat anti-mouse Ig antiserum coupled to horseradish peroxidase and tetramethyl benzidine was used as substrate. Optical density was read at 450 nm. Positive MAbs gave values of 0.3-> 1.0, values for negative MAbs were less than 0.05.

RESULTS Production and characterisation of 10

new

MAbs

Ten new MAbs were generated and characterized (Table 2). All 10 of the MAbs reacted in Western blot, 6 with the envelope precursor gpl60 and the external glycoprotein gpl20 and 4 with gp 160 and the truncated transmembrane protein gp32. All MAbs bound to envelope expressed on the surface of unfixed SIVinfected C8166 cells although the reactivities of one MAb to gpl20 (KK45) and 1 MAbs to gp32 (KK55) were weak. Three MAbs were IgG2a isotype and the remainder were all IgG,. Using predicted immunogenic peptides, 3 MAbs mapped to peptide 1003 (V2), 3 to peptide 1004 (V3), and 4 mapped to '3 peptide 954, the immunodominant region of gp41. One of the 10 new MAbs (KK54) had a significant neutralization titer of 800. The remaining 9 MAbs had low ( tenfold) than against a heterologous strain. Titers of the remaining 6 MAbs were not significantly different (< fourfold)

of

Seven Predicted Immunogenic Peptides

equivalent region

HIV-1

Sequence TMTGLKRDKTKE YNETWYSTC

RRPGNKTVLPVTIMSGLVFHSQPINDRPKQAWC QIINTWHKVGKNVYLPPREGC GGTSRNKRGVFVLGFLGFLAC

AIEKYLKDQAQLNAWGCAFRQVC QDPALPTREGKEGDGGEGGGNSSWPWQIEYIHFC LRRGGRWILAIPRRIRQGLEC

"The location of the amino acid sequence is based on the sequence of the

SIVmac25,

Reference

V2 V3 CD4 Binding region Fusion domain Gnann epitope

Kennedy epitope C gpl60

32H molecular clone pJ5

as

(9,10) (9,10) (11) (12) (13) (14) (15)

submitted to the

DDBJ, EMBO and Genbank under accession number D01065. The envelope protein has been assigned accession number JS0508 by the

Japan International Protein Information Database.

NEUTRALIZING EPITOPES ON SIV ENVELOPE

1149

Table 2. Characterization of Ten New Monoclonal Antibodies

to

Simian Immunodeficiency Virus

Mab

SFA

WB

RIPA

Isotype(IgG)

Neut. titer

KK43 KK52 KK54 KK42 KK45 KK46 KK39 KK41 KK53 KK55

+ ++ + +

+ + 160/120 + + 160/120 + + 160/120 + 160/120 ± 160/120 + 160/120

+ + +

1 2a 1 1 2a I 2a 1 1 1

30 80 800 60 SO 30

+

+ + + +

+

± 160/32 + 160/32

+ + + +

160/32 160/32

+

±

±

+

(SIV)

Peptide recognized 1003 1003 1003 1004 1004 1004 954 954 954 954

411

30

30

'

V2 V2 V2 V3 V3 V3

(Gnann) (Gnann) (Gnann) (Gnann)

Peptide mapping was determined using the predicted immunogenic peptides (Wellcome peptides). HIV-1 equivalent region; ++ strong reaction; ± weak reaction. Abbreviations: SFA, surface fluorescence; WB, Western blot; RIPA, radioimmune precipitation assay. a

=

against

=

the four SIV strains.

Only

one

=

MAb

against gpl20

(KK52) reacted with HIV-2 SBL6669 with a titer of 2.6. All four MAbs against the transmembrane protein cross-reacted with the HIV-2 strain with log10 titers of 2.7-3.4.

Peptide mapping When the 10 tested in ELISA

reactivity was

and 18 previously described MAbs were against the 7 predicted immunogenic peptides, seen with only three peptides, 1003 (V2), 1004 new

(V3), and 954 (Gnann) (Table 4). MAbs KK10, 13, 43, 52, and 54 reacted with the V2 peptide, KK42, 46, and 45 reacted with the V3 peptide, and KK14, 15, 16, 20, 39, 41, 53, and 55 reacted with the Gnann peptide. When gpl20 MAbs were tested the overlapping peptides all MAbs in the V2 group reacted with peptide 16 (aa 171-190). KK42 and 46 in the V3 group reacted with peptide 31 (aa321-330). KK45 in the same V3 group also reacted with peptide 31, but reactivity was greater with peptide 30 (aa311-330). In addition to these two groups of MAbs, KK3 and 18 reacted with peptide 1 (aa21-40) and KK.12 reacted with peptide 7 (aa81-100). When the gp41 MAbs were tested against peptides, overlapping epitopes within the Gnann region were identified. KK14 and 15 reacted with peptides 9 and 10 whereas the remaining 6 MAbs reacted only with peptide 10. Of the 28 MAbs, 5 have strong neutralizing activity and these

against

bind to two different sites. KK10 and KK54, in competition group 3, map to aal71-190 in the V2 region of SIV. Surprisingly, KK.13, 43, and 52 in the same competition group and mapping to the same V2 peptide did not show neutralizing activity. The second neutralizing site identified by MAbs KK5, 9, and 17 in competition group 1 could not be mapped with peptides. These MAbs also failed to react in immunoblot and did not react with recombinant fragments of env derived from Escherichia coli.7

DISCUSSION Published reports have described MAbs to SIV envelope which bind either the amino terminal half of gpl20 or to the transmembrane protein.6'7 The 10 new MAbs described herein include three to the V3 region of SIV which have not previously been reported and additional MAbs to V2 and Gnann epitopes. These MAbs together with 18 previously reported7 enabled us to define ten distinct binding sites and to establish their role in neutralization of SIV. Seven of the 10 sites were defined by overlapping peptides. Two sites are amino terminal to the VI region and one of these, identified by KK12, was shown previously to bind to aa88-96 of HIV-2. '7 One site is in V2, two related but distinct epitopes are

Table 3. Cross-Reactivity of Ten New Envelope Monoclonal Antibodies Against Four SIV Strains and HIV-2 Strain

Peptide specificity

Mab KK43 KK52 KK54 KK42 KK45 KK46 KK39 KK41 KK53 KK55 Results

V2 V2 V2 V3 V3 V3 Gnann Gnann Gnann Gnann are

SIVmac251

11/88

2.2 1.9 2.5 1.6 1.6 2.6 2.9 2.5

2.7 3.0

expressed as ELISA endpoint titers (log]0).

SIV.cynI86 2.5 2.6 2.6 3.4 1.7 3.0 3.1 3.3 3.0 3.2

siv„ 3.4 2.8 3.2 3.7 1.8 3.2 3.0 3.6 3.0 2.9

siv„

HIV-2 SBL

3.0 3. 1

2.6

3.0 3.6 1.7 3.1 2.8 3.4 2.7 2.7

2.7 3.4 3.0 3.0

KENT ET AL.

1150 Table 4. Reactivity

MAb

no.

(KK)

5, 9, 17a 8, 11, 19, 21, 22 10, 54, 13, 43, 52 3, 18

Neutralizing activity ++

+

of

Comp. group

45 7

5

14, 15

6

42, 46

with

Synthetic Peptides

Reactivity with predicted immunogenic peptides

1 2 3 4 ND ND ND

12

28 Envelope MAbs

16, 20, 39, 41, 53, 55

None None 1003 (V2) 1003 (V2) None None 1004 (V3) 1004 (V3)

None 954 (Gnann) 954 (Gnann)

Reactivity with overlapping peptides Residue

Peptide

None None 171-190 171-190 21-40 81-100 321-340 311-330 321-340 ND 591-610 601-620 601-620

None None EVA 774.16 EVA 774.16 EVA 774.1 EVA 774.7 EVA 774.31 EVA 774.30 EVA 774.31 ND EVA 798.9 EVA 798.10 EVA 798.10

no.

"Previously described as competition group 112. Recloning of this hybridoma yielded cells producing a neutralizing antibody with cross-reactivity with group 2. not determined. Comp. group; competition groups were defined using peroxidase- or biotin-labelled SKB MAbs raised ND against BK28 which identified 6 distinct competition groups; data reproduced from ref. 7. no

=

in V3, and two related sites were found in the Gnann region of gp41. The remaining three sites in competition groups 1, 2, and 5 could not be identified with peptides and may be discontinuous or conformational epitopes. The five MAbs within competition group 3 all map to the V2 region of gp 120 (aa 171 -190), but only two of these, KK 10 and KK54 show consistent virus neutralizing activity. All 5 MAbs are broadly cross-reactive with all SIV strains tested, but do not react with HIV-2 SBL. It is of interest that MAbs binding to the same 20 amino acid peptide have different biological activities. Mapping with smaller peptides will establish if two distinct epitopes exist within the amino acid sequence 171-190. The identification of MAbs to V3 of HIV-1 that protect against HIV-1 infection in chimpanzees and the ability of V3 peptides to absorb out the neutralizing activity from polyclonal sera suggest that the hypervariable V3 region may play a role in protection against HIV-1. In contrast, the SIV region equivalent to V3 in HIV-1 is genetically very conserved.,0,18 The 3 new MAbs to the SIV V3 region recognize two overlapping crossreactive epitopes but they do not have neutralizing activity. Furthermore, SIV peptides analogous to the HIV-1 V3 region are unable to elicit neutralizing antibody.19 These results sug¬ gest that the V3 region of SIV, unlike that of HIV-1, is not a significant linear neutralizing determinant. However, this does not mean that V3 is irrelevant in SIV vaccine protection. The V3 region in SIV may form part of a' conformational epitope which may play a role in CD4 binding' or cell fusion12 or another, as yet undefined, function. We have generated a number of MAbs to the transmembrane protein including 8 which react with the Gnann peptide but none of these have neutralizing activity. In contrast, the neutralizing antibodies induced by immunizing macaques with 4 synthetic peptides are believed to be directed against two immunodomi¬ nant regions of gp4120 one of which is equivalent to the Gnann epitope.'3 Furthermore, Kodama et al.6 described a strainspecific neutralization determinant at aal06-l 10 in gp41.

One of the sites which could not be identified with peptides is involved in neutralization of SIV. The neutralizing MAbs KK5, 9, and 17 in competition group 1 do not react with denatured antigen7 or linear peptides. KK5 and 17 react in ELISA with SIVmac251, SIVcynl86, and SIVsm7, but not SIVsmrnB670 suggesting that they recognize a cross-reactive epitope, whereas KK9 is strain specific and reacts only with SIVmac251.7 Although these three MAbs are in the same compe¬ tition group, this evidence suggests that more than one discon¬ tinuous or conformational epitope may exist. There is indepen¬ dent evidence that neutralization of SIV may require antibody to conformational epitopes of the external glycoprotein.19 Immu¬ nization of macaques with gpl 10 recombinant envelope protein elicited strong neutralizing activity. Furthermore, the neutraliz¬ ing activity in sera from infected macaques could be absorbed out with native gpl 20, but not with reduced or denatured gpl20, gpl40, or synthetic peptides. Immunization with the latter antigens was unable to elicit high titer neutralizing activity. Some neutralization determinants were probably cross-reactive because recombinant env from SIVmac25, absorbed the neutral¬ izing activity from sera of animals immunized with SIVsmmB670. The SIV neutralizing epitopes identified in this study recog¬ nize the V2 region or conformational or discontinuous epitopes. Similar epitopes are now being described for HIV-1.2'"25 Thus, although the V3 regions of SIV and HIV-1 may be functionally different other neutralizing sites may be analogous in the two viruses. Work must continue to define the biologically signifi¬ cant regions of SIV envelope and other viral proteins. Further analysis of MAbs recognizing conformational neutralizing epitopes will define more functional domains on SIV envelope and permit further comparisons with HIV-1. The relevance of these antibodies in protection may now be established by passive transfer into macaques and subsequent challenge with SIV. These studies will enhance the value of SIV vaccine experiments in macaques by enabling more reliable extrapolation to HIV-1 and the choice of appropriate strategies for AIDS vaccines.

clearly

1151

NEUTRALIZING EPITOPES ON SIV ENVELOPE the human immunodeficiency virus critical for interaction with the CD4

ACKNOWLEDGMENTS This work was funded in part by a grant from the Medical Research Council's AIDS Directed Program. We would like to thank Mr. R. Campbell for synthesis of the predicted immuno¬ genic peptides and would like to acknowledge Dr. H. Holmes and the EVA Reagent Program for commissioning and supply¬ ing the overlapping envelope peptides. We thank Sandra Gough and Lynn Plumb for final preparation of the manuscript.

985.

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1991;88:542-546.

2. Berman PW, Gregory TJ, Riddle L, Nakamura GR, Champe MA, Porter JP, Wurm FM, Hershberg RD, Cobb EK, and Eichberg JW: Protection of chimpanzees from infection by HIV-1 after vaccina¬ tion with recombinant glycoprotein gpl20 but not gpl60. Nature

1990;345:622-625. 3. Emini EA, Schleif WA, Nunberg JH, Conley AJ, Eda Y, Tokiyoshi S, Putney SD, Matsushita S, Cobb KE, Jett CM, Eichberg JW, and Murthy KK: Prevention of HIV-1 infection in chimpanzees by gpI20 V3 domain-specific monoclonal antibody. Nature

1992;355:728-730. 4. Hu S-L, Abrams K, Barber GN, Moran , Zarling JM, Langlois AJ, Kuller L, Morton WR, and Benveniste RE: Protection of macaques against SIV infection by subunit vaccines of SIV enve¬ lope glycoprotein gpl60. Science 1992;255:456-459. 5. Putkonen P, Thorstensson R, Ghavamzadeh L, Albert J, Hild K, Biberfeld G, and Norrby E: Prevention of HIV-2 and SIVsm infection by passive immunisation in cynomolgus monkeys. Nature

1991;352:436-438. 6. Kodama T, Burns DPW, Silva DP, Veronese FDM, and Desrosiers RC: Strain-specific neutralizing determinants in the transmembrane protein of simian immunodeficiency virus. J Virol 1991;65:20102018. 7. Kent KA, Gritz L, Stallard G, Cranage MP, Collignon C, Thiriart C, Corcoran T, Silvera , and Stott EJ: Production and characteri¬ sation of monoclonal antibodies to simian immunodeficiency virus envelope glycoproteins. AIDS 1991;5:829-836. 8. Kitchin PA, Cranage MP, Almond N, Barnard A, Baskerville A, Corcoran T, Fromholc C, Greenaway P, Grief C, Jenkins A, Kent K, Ling C, Mahon B, Mills K, Page M, Silvera P, Szotyori Z, Taffs F, and Stott EJ. Titration of SIVmac25I (32H isolate) in cynomolgus macaques for use as a challenge in vaccination studies. In: Animal Models in AIDS, H. Schellekens and M.C. Horzinek (eds.). Elsevier Science Publishers B.V. (Biomedicai Division), Amster¬ dam, 1990, pp. 115-129. 9. Modrow S, Hahn BH, Shaw GM, Gallo RC, Wong-Stahl F, and Wolf H: Computer-assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variable regions. J Virol

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M, Potz J, Basiripour L, Dorfman T, Goh WC, TerwilIiger E, Dayton A, Rosen C, Haseltine W, and Sodroski J: Functional regions of the envelope glycoprotein of human immun¬ odeficiency virus type 1. Science 1987;237:1351-1355. Gnann Jr JW, Nelson JA, and Oldstone MBA: Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus. J Virol 1987;61:2639-2641. Kennedy RC, Henkel RD, Pauletti D, Allan JS, Lee TH, Essex M, and Dreesman GR: Antiserum to a synthetic peptide recognizes the HTLV-III envelope glycoprotein. Science 1986;231:1556-1559. Chanh TC, Dreesman GR, Kanda P, Linette GP, Sparow JT, Ho DD, and Kennedy RC: Induction of anti-HIV neutralizing antibod¬ ies by synthetic peptides. EMBO J 1986;5:3065-3071. Hopp TP. and Woods KR: Prediction of protein antigenic determi¬

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Address

reprint requests to: Dr. Karen Kent NIBSC Blanche Lane South Mimms Potters Bar, Herts EN6 3QG England

Identification of two neutralizing and 8 non-neutralizing epitopes on simian immunodeficiency virus envelope using monoclonal antibodies.

Ten new monoclonal antibodies (MAbs) to SIV envelope were produced and characterized. Using a panel of 28 MAbs, 10 antibody binding sites on SIV envel...
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