AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 4, 1992 Mary Ann Liebert, Inc., Publishers

Monoclonal Antibodies to the C4 Region of Human Immunodeficiency Virus Type 1 gpl20: Use in Topological Analysis of a CD4 Binding Site JANE A. MCKEATING,1 JOHN P. MOORE,1 MORAG FERGUSON,2 HOWARD S. MARSDEN,3 SUSAN GRAHAM,3 JEFFREY W. ALMOND,4 DAVID J. EVANS,4 and ROBIN A. WEISS1

ABSTRACT We have raised antisera and monoclonal antibodies (MAbs) to the (4 region of HIV-1 gpl 20, using an antigen chimaera of poliovirus as immunogen. These MAbs and sera, together with MAbs to the same region raised by other methods, fall into three groups defined by their abilities to bind to recombinant gpl20 and/or the immunogenic peptide. In some cases, the amino acids recognized by the MAbs have been identified by pep-scan and by solution phase peptide inhibition of binding to recombinant gpl20. Our results indicate that the amino acids WQEVGKAMYA are exposed on the surface of recombinant gpl20. Antibodies to these amino acids on recombinant gpl 20 compete for soluble CD4 binding in vitro, but only weakly neutralize HIV.

INTRODUCTION

THE

SURFACE AND TRANSMEMBRANE

glycoproteins gpl20

of the human immunodeficiency virus type 1 (HIV-1) are encoded by the viral env gene and are essential structural components of the virion.I-S HIV binding to suscep¬ tible cells is mediated by gpl20, and the gpl20/gp41 complex is involved in subsequent fusion events leading to virus penetration into the cell (reviewed in Ref. 6). HIV-1, HIV-2, and simian immunodeficiency virus (SIV) infect predominantly cells ex¬ pressing the CD4 antigen, which is the principal receptor for gpl20.7~'(> The amino acids on CD4 that are involved, directly or otherwise, in contact with gpl20 have been delineated in considerable detail by antibody mapping studies, mutational analysis and crystallography."12 Much less is known, how¬ ever, about the corresponding amino acids in gpl20 that interact with CD4. Monoclonal antibodies (MAbs) and sera capable of blocking recombinant gp 120 binding to CD4 map to amino acid (aa) residues 413-456 in the fourth conserved (C4) region of gpl20.13-15 Deletion of 12 amino acids (426-437) from this region abolished CD4 binding.,3 Regions of gpl20 adjacent in and

gp41

the primary sequence'6 18 and others elsewhere in the envelope have also been implicated as important for interaction with CD4.15-'9-20 More recently, amino acids in the C2, C3, and C4 regions of gp 120 were shown by mutational analysis to influence CD4 binding21 and antibodies to a conserved, but unknown, conformationally sensitive region of gpl20 prevented CD4 binding.22 To determine which parts of the C4 region are exposed on the surface of native gpl20, we have performed a topological analysis with antibodies which we mapped when¬ ever possible to known amino acids. We have used a novel epitope presentation system, an antigen chimaera poliovirus expressing aa430-446 of gp 120 to generate some of the MAbs used in this study. The antigenic sites of poliovirus are well characterized23 and the three-dimensional structures of two serotypes of the virus have been determined at near atomic resolution.24·25 This detailed knowledge of the relationship between structure and antigenicity allowed the construction of intertypic antigen chimaeras of poliovirus,26 and the subsequent development of the Sabin 1 strain of poliovirus as an epitope expression vector.2728 The latter enables the replace¬ ment of antigenic site 1, a linear epitope protruding at the

'Chester Beatty Laboratories, The Institute of Cancer Research, Fulham Road, London SW3 6JB, England. 2National Institute for Biological Standards and Control, Potters Bar, Herts EN6 3QG, England. 'Institute of Virology, Church Street, Glasgow, Scotland. "•Department of Microbiology, University of Reading, London Road, Reading RG1 5AQ, England. 451

MCKEATING ET AL.

452

pentameric

apex of the icosahedral virus particle, with se¬ quences of choice. Such a system offers several advantages for epitope presentation: sequences are expressed in a known immunodominant position; and MAbs directed against the intro¬ duced epitope can be rapidly selected and screened by compar¬ ison with unmodified Sabin 1 virus. Our study shows that only a limited number of amino acids in the part of the C4 region that we analyzed are exposed on the surface of recombinant gpl 20. Binding of MAbs to these amino acids blocks binding of gpl20 to recombinant, soluble CD4 but the MAbs and antisera only weakly neutralize HIV.

MATERIALS AND METHODS Construction

of poliovirus antigen chimaeras

We constructed poliovirus antigen chimaera Sl/env/4 to present 17 amino acids from the C4 region of gpl20 in an immunodominant position on the surface of a poliovirus parti¬ cle. The HIV-1 amino acids included in this chimaera are

numbers 430-446 (NMWQEVGKAMYAPPISG) from the env gene sequence of HIV-1 (BRU), as catalogued by Myers et al.29 This numbering system includes the env leader peptide. Num¬ bering of other HIV sequences is as reported in the original publication: note that aa 430-446 in our numbering system corresponds to aa 409-425 in Ref. 13 and aa 425-441 in Ref. 21. To minimize confusion, we generally assign both amino acid single letter codes and numbers to sequences described in the text.

The cassette vector, pCASl, has been described in detail previously.27 It consists of a full-length Sabin 1 cDNA cloned in a pBR-derived plasmid, preceded by a T7 promoter, and modified to contain unique Sal-1 and Dra-1 restriction sites flanking the sequences encoding antigenic site 1. Complemen¬ tary oligonucleotides (5'-TC GAC AAC ATG TGG CAA GAG GTA GGT AAG GCA ATG TAC GCT CCA CCA ATT TCA GGT and 5'-ACC TGA AAT TGG TGG AGC GTA CAT TGC CTT ACC TAC CTC TTG CCA CAT GTT G) were annealed and ligated with Sal-l/Dra-1 digested pCASl. Recombinant plasmids were screened for the loss of the Dra-1 site, and the presence of the oligonucleotides confirmed by dideoxy DNA sequencing.30 RNA runoff transcripts were produced in vitro31 from the recombinant plasmid pSl/env/4 and used to transfect subconfluent Hep2C monolayers. A cytopathic effect was ob¬ served within 3 days and RNA extracted from the recovered virus (designated Sl/env/4) was sequenced32 to confirm the chimaeric nature of the genome. Using similar construction and recovery procedures, the antigen chimaeras Sl/env/9 (incorporating the gpl20 sequence APPISGQISCSSNID, aa 441-455) and Sl/env/10 (incorporat¬

ing LPCRIKQFINMWQEVG, aa 421-436) were generated. The gpl20 (BRU) sequences presented by these chimaeras flank and

partially overlap the sequence expressed by Sl/env/4.

Peptides and recombinant antigens peptides were prepared by standard procedures using an Applied Biosystems automated peptide synthesizer. Peptides Soluble

39

(DTITLPCRIKQIINMWQKVG,

aa

417-436) and 41 (KA-

MYAPPISGQIRCSSNITG, aa 437-456), overlapping 20-mers from the IIIB sequence, and peptide env4 (NMWQEVGKAMY¬ APPISG, aa 430-446) representing the 17 amino acids ex¬ pressed in poliovirus chimaera Sl/env/4, were provided by the MRC AIDS Directed Programme (ADP) reagent program. Overlapping decamer peptides from within the env4 sequence; PI (NMWQEVGKAM), P2 (WQEVGKAMYA), P3 (EVGKAMYAPP), P4 (GKAMYAPPIS), P5 (AMYAPPISGQ), and P6 (YAPPISGQIR) were donated by Dr. G.I. Evan (ICRF Labs, St. Bartholomew's Hospital, London), as was an irrelevant peptide, SRF-3 (HMMYPSPHAVMYAPTS). Peptides 730-34 (KQSSGGDPEIVTHSFNCGGE) and 740-42 (GQIRCSSNITGLLLTRDGGNS) representing aa 362-381 and aa 441-461 oftheC3 and C4 domains of gpl20 (IIIB) (numbered as in Ref. 21) were provided by the ADP reagent program. Immobilized hexameric and nonameric peptides were prepared by solid-phase chemistry for pep scanning essentially as described previously,33 except that the procedures were modified for Fmoc chemistry as recommended by the manufacturer (Cambridge Research Biochemicals, Harston, England). CHO-expressed gpl20 (Celltech Ltd., Slough, England) from the HTLVMIB strain of HIV-1 (BH10 clone) was obtained from the ADP reagent program, and has been described previ¬ ously.34 Soluble CD4 (sCD4) expressed in and purified from CHO cells35 was a gift from R.W. Sweet (Smith Kline Beecham, King of Prussia, PA). Antisera and monoclonal antibodies Rabbit antisera RIO, Rll, R12, R19, R20, R21 to the poliovirus chimaera Sl/env/4; R213, R214, R215 to chimaera Sl/env/9; and R228, R229, R230 to chimaera Sl/env/10 were raised against sucrose-purified virus as described previously.28 Immunization was in Complete Freund's Adjuvant (CFA) and sequential bleeds were taken every month for three months. Antisera to ovalbumin-conjugated peptides 740-34 and 740-42 in CFA were raised in three different rats by standard proce¬ dures, and were gifts from J Cordell and C Dean (ICR Sutton). Eleven monoclonal antibodies to the Sl/env/4 chimaera were prepared as described previously.28·36 These were designated

1662, 1663, 1664, 1697, 1791, 1792, 1794, 1795, 1804, 1807,

and 1808. All these MAbs were specific for at least part of the HIV-1 insert, as they did not react with the parental Sabin 1 poliovirus (data not shown). MAbs 536 and 537 raised against gpl2014 were generously donated by DD Ho. MAbs ICR38.8f and ICR38. la to recombinant gpl2034 were gifts from J Cordell and C Dean (Institute of Cancer Research, Sutton).37

Peptide binding assay Microtiter plates (Immulon-II, Dynatech Ltd., Billinghurst, England) were coated overnight with peptide at 10 µg/ml (100 µ /well) in 100 mM sodium hydrogen carbonate buffer, pH 8.6. After washing twice with 144 mM NaCl, 25 mM Tris, pH 7.5 (TBS), and blocking with 2% nonfat milk powder (Marvel, Cadbury Ltd.) in TBS for 1 h, sera or MAbs (100 µ ) diluted in TBS/2% Marvel was added for 1 h at room tempera¬ ture. After three washes with TBS, bound antibodies were detected with alkaline phosphatase (AP) -labelled antispecies

immunoglobulin (Dakopatts Ltd., High Wycombe, England)

gpl20

453

and the AMPAK system (Novo Nordisk, Cambridge, England) as described previously.36 ELIS As using pin-bound hexameric and nonameric peptides were performed using procedures rec¬ ommended by the manufacturers (Cambridge Research Bio-

poliovirus reagents and polyclonal anti-gpl20 serum were car¬

EPITOPE MAPPING THE C4 REGION OF HIV-1

chemicals).

Binding of antibodies

to

gpl20: competition

with sCD4

Binding of antibodies to immobilized gpl20 was assessed by ELISA essentially as described previously.34'38 The capture antibody was sheep antiserum D7324 to the C-terminal 18 amino acids of gpl20, and gpl20 immobilized on the solid phase by this antibody binds sCD4 with high affinity.34"38 Unless speci¬ fied otherwise, no detergent was present at any stage of the assay. All binding and competition studies were carried out in a buffer comprising 2% nonfat milk powder (Cadbury's Marvel) and 20% sheep serum (Seralab, Crawley) to minimize nonspe¬ cific binding. To confirm that the binding of MAbs to gp 120 was not compromised by prior capture of gpl 20 onto the solid phase via antibody D7324, titration curves of MAb and gpl20 were also performed in solution before precipitation of the complexes onto D7324. Similar data were obtained by either method. In some experiments sera, MAbs or sCD4 were tested for compe¬ tition with rabbit Sl/env/4 antisera (or other sera or MAbs) for binding to gpl20 by incubation for 1-2 h at room temperature with gpl20 (2.5 nM) in TBS/2% Marvel/20% sheep serum (100 µ ) prior to addition of Sl/env/4 antiserum (10 µ ) for a further 1 h. In some experiments the order of addition of Sl/env/4 antisera and competitor was reversed.39 gpl20 was then captured onto adsorbed D7324 antibody, and bound rabbit antibodies detected with SaRablg-AP and AMPAK. The effect of sera or MAbs on the binding of sCD4 to immobilized gp 120 was determined essentially as described elsewhere.34"39

Antigen blocking and neutralization

C8166.40

RESULTS

of the poliovirus

chimaera Sl/env/4

Monoclonal antibodies specific for antigenic site 1 of Sabin 1 did not react with S l/env/4 in antigen blocking or neutralization assays.36 However, the reactivity of the chimaera with poly¬ clonal Sabin I antiserum, or MAbs specific for antigenic sites 2 and 3,23 was unaffected by the extensive modification of antigenic site 1. Polyclonal sheep antiserum (ADP 401) to recombinant gpl 20 neutralized 100 TCID50 units of Sl/env/4 at a titer of 1:5760. The HIV-1 neutralizing MAbs 536, 537,14 and ICR38.1a, ICR38.8Í37 neutralized Sl/env/4 at 0.62, 0.62, 0.009, and 0.009 µg/ml, respectively, but did not neutralize wild-type Sabin 1. These MAbs are specific for gpl20 amino acids NMWQEVGKAMYAPPISG,14'37 suggesting that the conformation adopted by the gpl20-derived sequence engi¬ neered onto the surface of S l/env/4 was comparable to that in the native glycoprotein. Similar neutralization analyses with anti-

on

chimaeras Sl/env/9 and Sl/env/10 to confirm the

expression of their inserted gp 120 epitopes (C .Velia and Ferguson, unpublished data). These studies prompted us to

M. raise antisera and MAbs to the chimaeras.

Binding of antisera region

and MAbs

to

peptides from

the C4

We used peptides from the C4 region to compare the reactiv¬ ities of S l/env/4 antisera and MAbs with those of other MAbs raised to different immunogens. Final bleed antisera from all five rabbits (RIO, Rll, R12, R19, R20) immunized with the Sl/env/4 chimaera bound to peptide env4 at dilutions ranging from 1:800 to 1:11,800, whereas there was no significant binding to the irrelevant peptide SRF-3 (Table la). Thus there was an efficient humoral immune response to the gpl20 epitope incorporated into the Sl/env/4 chimaera. Rabbit antisera R214 and R215 to Sl/env/9 bound to peptide 41 which included the gpl20 sequence expressed on the chimaera (Table 1, legend). However, antisera to Sl/env/10 failed to bind to peptide 39, the closest available peptide to the inserted epitope. Rat antisera to ovalbumin-conjugated peptides (740-34 and 740-42) from the

Table 1

Binding of Sl/env/4 Antisera env-4 Peptide and gpl20

and

MAbs

Reciprocal dilution giving half-maximal binding to: Antiserum

env-4

(a) RIO

3,000 11,800

assays

Poliovirus neutralization and antigen blocking assays were carried out as described elsewhere,36 and HIV neutralization was assessed by inhibition of infection of the CD4+ cell line,

Characterization

ried out

correct

(b)

Rll R12 R19 R20 R21 MAb 1662 1663 1664 1697 1794 1804 1807 1808 1795 536 537 ICR38.1a ICR38.8f 1791 1792

800

Group (A) (A) (A) (A) (A) (A) (A) (A) (B) (B) (B) (B) (B) (C) (C)

gpl20 100 500 100

11,000 5,800

9,600