AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 6, Number 11, 1990 Mary Ann Liebert, Inc., Publishers

Enhancement of Soluble CD4-Mediated HIV Neutralization and gpl20 Binding by CD4 Autoantibodies and Monoclonal Antibodies JOHN P.

MOORE,1 QUENTIN J. SATTENTAU,2 and PAUL R. CLAPHAM1

ABSTRACT We have identified 6 sera containing autoantibodies to CD4 in 174 human immunodeficiency virus-type (HIV-1) positive sera tested in an antigen-capture enzyme-linked immunosorbent assay (ELISA) using sCD4, and none in 34 HIV type 2 sera. These autoantibodies do not bind to cellular CD4, but react with sCD4 to increase its binding in ELISA to monoclonal antibodies and the HIV surface glycoprotein gpl20. The effect of CD4 autoantibodies is mimicked by monoclonal antibodies to the third and fourth domains of CD4. The enhanced sCD4 binding to gpl20 in ELISA is reflected by a reduction in the concentration of sCD4 required to neutralize HIV-1 and HIV-2 infection in tissue culture when CD4 autoantibodies or the relevant monoclonal antibodies were

present.

INTRODUCTION

THE(HIV-1

HUMAN IMMUNODEFICIENCY VIRUSES types 1 and and HIV-2) bind to the cell surface via a

binding of CD4 to gpl20 was HIV+ 2

high affinity interaction between the viral surface glycoprotein gpl20 and the cellular CD4 glycoprotein.1-5 CD4 is the principal cellular receptor for HIV, although there are alternative routes of infection that are CD4-independent.6"8 Humans do not normally

make antibodies to their own CD4 molecules, which are recognized as "self by the human immune system. However, autoantibodies to CD4 are found in the sera of a proportion (—5-10%) of HIV-infected individuals.9-12 The origin of these autoantibodies is uncertain, but they do not appear to be idioty pic and do not react with the gp 120-binding site on domain 1 of CD4. No prognostic or diagnostic significance of autoantibodies to CD4 has been found.9-12 A promising approach to immunotherapy against HIV infection is the use of CD4 made soluble by truncation before its plasma membrane insertion site (sCD4) as an inhibitor of the virus-CD4 interaction.13-17 sCD4 has been shown to inhibit infection of CD4 cells by a range of divergent HIV-1 and HIV-2 isolates in vitro.6'13-17 We were interested to see how the

affected by antibodies to CD4. therefore screened for autoantibodies that sCD4 in an antigen-capture enzyme-linked immu-

sera were

bound to nosorbent assay (ELISA), and these sera were then tested in an ELISA that measures the gpl20-sCD4 interaction.1819 Six of 174 (3.6%) HIV-1+ sera, but none of 34 HIV-2+ sera, contained antibodies that recognized sCD4. The three sera with the highest titer of CD4 autoantibodies increased the binding of sCD4 to gpl20 by approximately threefold, and this effect was also seen with monoclonal antibodies (MAb) mapping to domains 3 and 4 of sCD4. The same sera and M Abs also potentiated the neutralizing action of sCD4 on HIV-1 and HIV-2 infection of CD4+ cells in vitro, reducing the concentration of sCD4 required for inhibition by approximately 4-8-fold.

METHODS Sera sera from HIV-1-infected individuals (U.K. screened for anti-CD4 antibodies as described

A total of 174

origin)

were

'Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, England. 2Academic Department of Genito-Urinary Medicine, University College and Middlesex School of Medicine, James Pringle House, 73-75 Charlotte Street, London W1P 72D, England. 1273

MOORE ET AL.

1274

below. Six of these were positive: QC1 was from an anonymous male homosexual (Dr. J.N. Weber, Royal Postgraduate Medical School, Hammersmith Hospital, London) and was from a panel of sera that has been described previously;18-20 L-708 was from an asymptomatic male homosexual negative for p24 antigen (from Dr. E.A.C. Follett, Ruchill Hospital, Glasgow, Scotland); QS-43, QS-53, QS-101, and QS-123 were from a panel of sera from male homosexuals, and were provided by Dr. C. Loveday, University College and Middlesex Hospital Medical School. A selection of 34 HIV-2 sera from a WHO panel was obtained from the MRC AIDS Directed Programme (ADP) Resources Programme. Eight were from Senegal, 5 from the Ivory Coast, and 21 from Guinea Bisseau. CBL-JA, a rabbit polyclonal antiserum raised against sCD4,16 was prepared by M. Marsh (Chester Beatty Labs).

Antibodies

BioReagents, Dublin, Ireland) is an affinitypolyclonal antibody to a peptide APTKAKRRVsheep purified VQREKR from the C-terminus of gpl20 (IIIB) that is highly conserved between sequenced HIV-1 isolates.21 The alkaline phosphatase conjugate of D7324 (D7324-AP) was prepared by Novo Nordisk (Cambridge, England) and is available from Dr. H. Holmes, ADP Resource Programme Manager, NIBSC, Potters Bar, England. OKT4A (Ortho Diagnostics Inc., Raritan, NJ) is a murine M Ab that maps to domain 1 of CD4 and blocks gpl20 binding and HIV infection.1-2-22-23 MAbs OKT4 (Ortho Diagnostics), MT429 (P Reiber and G Reithmuller, Munich University), L120 (D. Buck, Becton Dickinson Monoclonal Antibody Center, San Jose, CA) Q425, Q428, and Q453 all map to epitopes within domains 3 and 4 of CD4 (24 and unpublished data). MAbs L83 and LI88 (D. Buck) recognize epitopes in domains 1 and 2 of CD4.25 Q425 and Q428, but not the other MAbs, block HIV infection at a stage subsequent to CD4 binding.24

Cadbury Ltd) in TBS. Pairs of wells were incubated with or without 1 pg/ml sCD4 (CHO) in 2% Marvel/TBS for 2 h, then unbound sCD4 was removed by washing twice with TBS. Sera were added as 1:500 dilutions in 100 p.1 of TMT/SS (20% sheep serum (Seralab, Crawley, England), 2% Marvel, 0.5% Tween-20 in TBS) to wells containing or lacking sCD4 and incubated for 1 h. Unbound serum was removed by washing four times with 200 u.1 of AMPAK wash buffer (Novo Nordisk, Cambridge) and bound antibodies detected with alkaline phosphatase-conjugated goat antihuman IgG-AP (1:1000 in TMT/ SS) (Seralab) for 1 h. Some batches of this conjugate crossreacted significantly with murine IgG: the one used in the studies described below did not. Bound alkaline phosphatase was measured with the AMPAK ELISA amplification system (Novo

Nordisk) as previously described.26-28

A serum was designated as positive for autoantibodies for sCD4 if the OD492 signal in the presence of sCD4 was reproducibly at least 0.200 greater than that in the absence of sCD4. Positive sera were then titrated (see below).

D7324 (Aalto

Recombinant gpl20 and sCD4 sCD4 prepared in and purified from Chinese hamster ovary (CHO) cells was a gift from R.W. Sweet (Smith Kline and French Laboratories, King of Prussia, PA) and has been described previously.16 The combined third and fourth domains of sCD4 were prepared by limited proteolysis of sCD4 and subse-

quent purification.24

Recombinant gpl20 from CHO cells (Celltech Ltd, Slough, England) were obtained from the ADP Resources Programme. Some properties of this gpl20 preparation have been re-

ported.1819

Screening for CD4 autoantibodies in human sera OKT4 (or in some experiments, OKT4A or LI20) was adsorbed to Immulon 2 microtiter plates (M129B, Dynatech) by incubation overnight at 2.5 pg/ml in 100 pi of 30 mM NaHC03, pH 8.5. Unbound antibody was removed by washing twice with TBS (144 mM NaCl, 25 mM Tris, pH 7.5) and the plates were blocked for 30 min with 2% nonfat dry milk powder (Marvel,

Assay for gpl20 binding effect of CD4 antibodies

to

sCD4:

The method has been described in detail elsewhere.18,19 Briefly, MAb OKT4 was adsorbed to Immulon 2 plates, the plates were washed and blocked with TBS/2% Marvel and sCD4 (1 u-g/ml) added in 100 u.1 of the same solution. After 2 h, the wells were washed twice with TBS, incubated for 1 h with TBS/2% Marvel/20% sheep serum containing HIV+ serum or MAb, then washed twice with TBS. Next, gpl20 was added at different concentrations in 100 p.1 of TBS/2% Marvel for 2 h, then unbound gpl20 was removed by washing twice with TBS. Bound gpl20 was detected with D7324-AP (1:1000,0.5 u.g/ml) in TMT/SS, and AMPAK.

Assay for sCD4 binding to OKT4: effect of CD4 antibodies The assay was as described above, except that sCD4 was added at a range of concentrations in TBS/2% Marvel/20% sheep serum with or without HIV+ serum or MAb. After 2 h, unbound sera and sCD4 were removed by washing twice with TBS and bound sCD4 detected with gpl20 (1 p,g/ml, 8.3 nM) and D7324-AP as above. Note that in the ELISA experiments, incubations were carried out in the presence of 20% normal sheep serum to control for effects attributable to nonspecific

immunoglobulins.

Inhibition

of HIV infection by sCD4

Inhibition of sCD4 of HIV infection of C8166 cells was assessed as described previously,6 except that a range of concentrations of sCD4 was mixed with sera (1:1000) or MAbs (3 (xg/ml) before addition to the cells. The inhibitory titer was assessed as the sCD4 concentration that inhibited syncytia formation by 98% in 3 of 3 wells. At the 1:1000 dilution used, none of the sera was neutralizing for HIV-1 or HIV-2. None of the CD4 MAbs was neutralizing except for Q425 and Q428.24

CD4 AUTOANTIBODIES AND MAbS ENHANCE

gpl20-sCD4 BINDING AND HIV NEUTRALIZATION

RESULTS CD4 autoantibodies in human sera We screened HIV+ sera for antibodies to CD4 using an antigen capture assay in which sCD4 was immobilized on the

solid phase via OKT4 antibody. This format was chosen rather than binding sCD4 directly onto the plate surface [e.g., 9-11] because we were interested in anti-CD4 antibodies that modulated the gpl20-sCD4 interaction. In our hands, sCD4 bound directly'8to a plate surface is denatured and unable to bind gp 120, whereas sCD4 bound via OKT4 binds gp 120 with high affinity.1819 The OKT4 MAb recognizes the third and fourth domains of sCD4 and does not modulate the gpl20-CD4

interaction.22

178-369

(domains 3 and 4) of sCD4, using L120

1275 as

capture

of the sera recognized the third and fourth domains, but to a variable extent. As a percentage of antibody binding to sCD4, binding to the third and fourth domains was:

antibody. Each

QC1, 45%; L-708, 33%; QS-43, 80%; QS-53, 75%; QS-101, 40%; QS-123, 20%.

Fluorescence-activated cell sorter (FACS) analysis was used to assess whether the autoantibodies present in HIV+ sera bound to CD4 on the surface of SupTl T cells or normal human

blood lymphocytes (PBLs). None of the 6 sCD4+ could bind to cellular CD4 in this assay, under conditions where OKT4 binding was clearly detected (data not shown). Thus, although the autoantibodies were raised against CD4 of cellular origin in HIV-infected individuals, they are unable to bind to native CD4 in vitro. Similar data have been reported

peripheral sera

Screening 174 HIV-1+ sera revealed 6 that reacted significantly with sCD4 in the antigen-capture ELISA, a prevalence of previously.10'12 3.4%. None of 34 HIV-2+ sera was positive for anti-sCD4

antibodies under the same conditions. When 160 of the HIV-1 sera were rescreened using OKT4A as capture antibody, no additional CD4 autoantibody-positive sera were identified. Titration of the six positive sera showed that the CD4 autoantibody titers were variable (Fig. la). The most potent serum, QC1, had a midpoint titer of 9,000 (reciprocal dilution giving 50% binding). The corresponding titers for the other sera were: L-708, 3,000; QS-101, 2,000; QS-43, QS-53, and QS-123, each approximately 400. It has been reported that CD4 autoantibodies in patient sera are unable to precipitate a truncated sCD4 comprising the 182 amino-terminal amino acids, ' ' implying recognition by the sera of sequences in the carboxy-terminal third and fourth domains. We tested this by comparing the binding of the 6 CD4 autoantibody-positive sera to sCD4 and to a peptide spanning residues

FIG. 1. Detection of autoantibodies to CD4 in HIV+ sera. Sera at the concentrations indicated were added to wells containing sCD4 captured onto the solid phase by OKT4 and bound human antibodies detected. In the absence of sCD4 none of the sera gave significant OD492 signals (> 0.100) at any dilution tested. QC1 (•), L-708 ( ), QS-43 (a), QS-53 ( ), QA-101

(o), QS-123 (ü), QC5 ( a) (typical CD4-, HIV+ serum).

Effect of CD4 autoantibodies on the binding of sCD4 to OKT4 and gpl20 in ELISA ELIS As for monitoring gpl20 binding to sCD4 and for analyzing the effect of HIV+ sera on this interaction have been

one of these assays, sCD4 is immobilized solid indirectly phase via adsorbed OKT4 antibody, gpl20 is reacted with sCD4, and bound gpl20 detected with an anti-gpl20 antibody. In this and other assays the gpl20-CD4 interaction is blocked by anti-gpl20 antibodies in HIV+ serum,18'29-30 which we have attributed in part to antibody cross-linking of gpl20 into immune complexes.18 However, it was noted that low concentrations of serum QC1 enhanced the binding of gpl20 to sCD4 in ELISA.18 To analyze this effect, the binding of sCD4 to OKT4 and of gpl20 to sCD4 was measured in the presence and absence of CD4+ and CD4~ sera (Fig. 2, Table 1). Note that in all the experiments described below, binding of gpl20 to the solid phase was always completely dependent on the presence of sCD4 and was inhibited by OKT4A, indicating that the effects of the CD4+ sera were not attributable to gpl20 binding to any anti-gpl20 antibodies adsorbed nonspecifically to the solid phase. Furthermore, similar data were obtained when sCD4 was bound to the solid phase via MAb L120 instead of OKT4 (not shown), demonstrating that the effects were not artefacts of OKT4 usage. sCD4 bound half-maximally to OKT4 at 1.0 ± 0.2 nM. However, the half-maximal sCD4 concentration was reduced 11-fold to 0.093 ± 0.012 nM by the simultaneous addition of CD4+ serum QC1 (Fig. 2a). The other 5 CD4 antibody-positive sera also increased the binding of sCD4 to OKT4, as did the rabbit anti-sCD4 serum CBL-JA (Table 1). CD4" sera were without effect (e.g., QC6 in Fig. 2a and data not shown). These experiments clearly demonstrate that the CD4 autoantibodies bind to sCD4 in solution, in contrast to a previous report.12 The binding of gp 120 to sCD4 with or without prebound CD4 autoantibodies was then studied (Fig. 2b). In the absence of serum, gpl20 bound half-maximally to sCD4 at 3.2 ± 0.3 nM, but when QC 1 serum was bound onto OKT4-immobilized sCD4 and excess serum was washed away prior to gpl20 addition, the gpl20 concentration giving half-maximal binding was reduced 3.3-fold to 0.96 ± 0.05 nM. When QC1 serum (1:1000) was added simultaneously with sCD4 during the binding reaction with OKT4, the concentration of gpl20 required subsequently

described.1819 In on a

1276

MOORE ET AL. Table 1. Effect of CD4+ Sera on the Binding of sCD4 TO OKT4 and gpl20

[sCD4]-OKT4 (nM)

°°«92

Serum None

QC1

L-708

QS-43 QS-53 QS-101 QS-123

CBL-JA

0

3) or means ± SD (n < 3), and are the concentrations in nM giving half-maximal binding in the presence or absence of the sera indicated (1:1000 dilution). The sera were added to sCD4 after its binding to OKT4 as in Fig. 2b. Column 1 : binding of sCD4 to OKT4; Column 2: binding of gpl20 to sCD4.

0°482



«,—8

»i==v>° 0

0-05 0-1

»120 (KM)

FIG. 2. Effect of CD4 autoantibodies on binding of sCD4 to 0KT4 and gpl 20. (a) sCD4 at the concentrations indicated was incubated with immobilised 0KT4 together with no added serum (o) or with 1:1000 dilutions of sera QC1 (•) or QC6 ( ). After washing away unbound sCD4 and sera, bound sCD4 was detected with a saturating concentration of gp 120 (1 u-g/ml, 8.3 nM) and D7324-AP. (b) sCD4 (1 p,g/ml; 22 nM) was incubated with immobilized OKT4. After washing away unbound sCD4, no serum (o) or 1:1000 dilutions of sera QC 1 (•) or QC5 ( ) or a HIV-2+, CD4- serum ( ) was added. After washing away unbound serum, gpl20 was added at the concentrations indicated, and bound gpl20 detected with D7324-AP.

for half-maximal binding to sCD4 was reduced by 7-fold to 0.45 ± 0.07 nM (n 5), and a similar effect was seen with serum L-708 (data not shown). Consistent with this, an 8-fold higher concentration of MAb OKT4A was required for 50% inhibition of gpl 20 binding to sCD4 under the latter conditions than when QC 1 serum was not added (data not shown). Note that in neither assay was CD4+ serum present in solution when gpl20 was added; only antibodies bound to sCD4 remain at this stage of the assay, so anti-gpl20 antibodies that potentially inhibit the sCD4-gpl20 interaction18 do not interfere. Of the other five CD4 + human sera tested, L-708 and QS-101 also increased the binding of gpl20 to sCD4, whereas QS-43, QS-53, and QS-101, which contain approximately 10-fold =

lower concentrations of CD4 autoantibodies, had no significant effect at dilutions of 1:1000 (Table 1). Rabbit anti-sCD4 serum CBL-JA did not enhance gpl20-sCD4 binding, probably because this serum also contains antibodies to the VI domain which antagonize gpl 20 binding. None of five HIV-1+ or three HIV-2+ CD4- human sera at 1:1000 dilutions had any enhancing effect on the binding of sCD4 to OKT4 or of gp 120 to sCD4 (Fig. 2 and data not shown), indicating that the enhancing effect seen with sera QC1, L-708, and QS-101 was attributable to anti-CD4 antibodies.

Effect of monoclonal antibodies on the binding of gpl20 to sCD4 Since

a

proportion of the CD4 autoantibodies in patient sera

the third and fourth domains, we tested whether monoclonal antibodies to these domains could increase the affinity of gp 120 for sCD4 in ELISA. All four of the third/fourth domain M Abs tested reduced the concentrations of gpl 20 required for half-maximal binding to sCD4 by 3-6-fold (Table 2), which is similar to the effect of QC1 serum. However, mabs L83 and LI88 to the first and second domains of sCD4 had no effect. Four of the five MAbs tested also increased the binding of sCD4 to OKT4 (Table 2).

recognize

Effect of CD4 autoantibodies and MAbs on by sCD4 of HIV infection in vitro

inhibition

sCD4 is a potent inhibitor of HIV infection in vitro.6- '3-17 We tested whether its neutralizing action could be increased by HIV+ sera containing CD4 autoantibodies, or by MAbs to the third and fourth domains of CD4, that increased the binding of gpl20 to sCD4 in ELISA. In an initial experiment the concentration of sCD4 required to block 98% of syncytium formation by the HIV-1 isolate HTLV-IIIB was reduced by 2-8-fold by a 1:1000 dilution of several sera containing CD4 autoantibodies. (Higher concentrations of these HIV+ sera could not be used because of interference with the sCD4 neutralization assay by

CD4 AUTOANTIBODIES AND MAbS ENHANCE of CD4 MAbS on to OKT4 and

Table 2. Effect

gpl20-sCD4 BINDING

Domain

Expt.

1 None L83 L188

Q425 Q428 Q453 QC1 serum Expt. 2

L2 1,2 3 3 3

None

Q425 Q428

3 3

MT429

QC1

3,4

serum

1277

Table 3. Effect of HIV+ Sera on Neutralization of HIV-1 and HIV-2bysCD4

Binding of sCD4

gpl20

MAb

AND HIV NEUTRALIZATION

[sCD4]-OKT4 (nM)

[gpl20]-sCD4

1.1 0.6 1.1 0.4 0.3 0.3 0.06

2.7 2.7 2.7 0.7 0.9 1.2 0.9

(nM)

nM sCD4

Serum

HTLV-IUb

HTLV-IIIrf

LAV-2IROD

None

6.9 0.42 3.4 6.9 6.9 1.7 1.7 6.9

6.9 1.7 6.9 6.9 6.9 6.9 6.9 13.8

110 27 110 110 110 220 220 220

QCP QC2 QC3 QC4 QC5 QC6 QC7

1.2 0.26 0.26 0.21 0.24

required for neutralization

Data

the concentrations of sCD4 required to inhibit formation by greater than 98% in 3/3 cultures infected with the HIV strains indicated. Sera were present at 1:1000 dilutions. aQCl is positive for CD4 autoantibodies, and QC7 is a typical HIV- control serum. are

syncytia

Data are the concentrations in nM giving half-maximal binding in the presence or absence of the mabs indicated (3jag/ml). QC1 serum (1:1000 dilution) was included for comparison. In column 1 is shown the domains recognized by the MAbs; Column 2: binding of sCD4 to OKT4; Column 3: binding of gpl20 to sCD4. Note that in Experiment 2 a different stock of recombinant CHO gpl20 with a slightly higher affinity for sCD4 was used.

the gpl20-sCD4 interaction in ELISA, or on neutralization by sCD4. MAbs Q425 and Q428 were not tested in this assay because they are capable of neutralizing HIV-1 in their own

right.24

DISCUSSION antibodies present.) The inhibitory sCD4 concentrations were (nM): No serum, 28; QC1, 3.5;

neutralizing anti-gpl20

L-708, 7.0; QS-43, 14; QS-53, 7-14; QS-101, 7; QS-123, not tested. Thus the sera having the greatest effect were those with the higher CD4 autoantibody titers (QC1, L-708, QS-101) and

vice versa. In the above experiment, none of three HIV+, CD4 sera (1:1000 dilution) reduced the inhibitory sCD4 concentration by more than twofold. We consider a twofold shift either way in the sCD4 neutralization titer to be within the experimental error of the assay. Discrimination between HIV+ sera that were positive and negative for CD4 autoantibodies was then studied in more detail (Table 3). In the first experiment, three of the CD4 sera (QC2, QC5, QC6) enhanced sCD4 neutralization of HTLV-IIIB by 2-4-fold, whereas the CD4+ QC1 serum enhanced it by 16-fold. The weak effect of the three CD4-, HIV-1+ sera is attributable in part to experimental variation in the assay and perhaps partially to synergy between sCD4 and subthreshold concentrations of the neutralising anti-gpl20 antibodies that are present at relatively high titers in these sera.18'20 However, none of the CD4- sera had any effect on sCD4 neutralization of HTLV-IIIRF, but QC1 reduced the neutralizing sCD4 concentration by 4-fold. Finally, only QC1 enhanced the neutralizing effect of sCD4 on HIV-2 (LAV-2/ROD) infection "

-

(by 4-fold).

MAbs to the third and fourth domains of CD4 that increased gpl20 binding to sCD4 in ELISA were then tested for their ability to enhance the neutralizing effect of sCD4 on HIV-1 (HTLV-IIIB) infection (Table 4). MAbs MT429 and LI20 reduced the neutralizing sCD4 concentration by 2-4-fold, whereas the control MAbs L83 and L188, had no effect either on

We have identified sera that contain autoantibodies to CD4 in 3.4% of the 176 HIV-1+ sera and none of the 34 HIV-2+ sera tested in an antigenic capture ELISA. The prevalence of CD4 autoantibodies we found is lower than previous reports of 32/253, (12.6%);9 3/33, (9.1%),10 9/91 (9.9%);n and 5/97 (5.2%).12 Although it is possible that capturing sCD4 via OKT4 prevents detection of autoantibodies that bind to epitopes spatially adjacent to the OKT4 epitope on the third and fourth domains, we did not detect additional CD4+ sera when sCD4 was captured onto the solid phase via the OKT4 A epitope on V1. The higher prevalence of CD4 autoantibodies reported previously9-12 may, therefore, be due to the recognition by some sera of epitopes on sCD4 that are exposed only when the

Table 4. Effect of CD4 MAbs on Neutralization of HIV-1 BYsCD4

MAb None MT429 L120 L83 L188

Domain

3,4 4

1,2 1,2

nM sCD4 required for neutralization 55 13.8 13.8 55 55

Data are the concentrations of sCD4 required to neutralize or absence of the CD4 MAbs

HTLV-IIIrf in the presence indicated (3 jug/ml).

1278

molecule is denatured by direct absorption on a plate surface. The use of sera from a different set of HIV+ individuals may also be a contributory factor. A variable proportion of the CD4 autoantibodies were to epitopes on the third and fourth domains of sCD4: in some sera antibodies to these domains predominated, whereas in others they were a minority. It is possible that epitopes required by these sera may be altered or lost when the third and fourth domains are proteolytically cleaved from sCD4. Our data therefore neither support nor unambiguously refute the contention1} that only epitopes in the third and fourth domains of sCD4 are recognized by patient sera. None of the sera we identified as positive for CD4 autoantibodies by binding to sCD4 contained antibodies that bound to the surface of CD4+ cells. This is consistent with the findings of Chams et al.'° We do, however, clearly demonstrate that CD4 autoantibodies bind to sCD4 in solution and not just when sCD4 is denatured by absorption on a solid phase. This contrasts with the report of Wilks et al.12 Indeed, the effect of CD4 autoantibodies on the subsequent binding of sCD4 to gpl20 is greater when sCD4 and the sera are mixed in solution than when the sera are added to OKT4-immobilized sCD4. This may indicate better exposure of the relevant sCD4 epitopes in solution. The origin of CD4 autoantibodies is uncertain, but plausible explanations are that they arise as a result of complexing of gpl20 with CD4 rendering CD4 immunogenic to the human immune system, or from degraded CD4 exposed to the immune system when CD4+ cells are killed by HIV infection.9-11 Consequently, the epitopes recognized by the antibodies may be cryptic on cellular CD4 and exposed only when gpl20 is bound or when the structure of CD4 is perturbed in some other way (e.g., truncation to sCD4, enhanced in some instances by direct absorption of the molecule to a plastic surface). HIV+ sera containing CD4 autoantibodies, but not sera lacking these antibodies, and some CD4 MAbs increased the binding of sCD4 to OKT4 and gpl20 in ELISA. The former effect (Fig. 2a) is not likely to be of physiological relevance and is most probably predominantly an ELISA artefact due to crosslinking of sCD4 into immune complexes. Binding of such complexes to immobilized OKT4 would increase the capacity of the ELISA wells for sCD4 at each sCD4 input concentration, and hence the amount of gpl 20 that can be bound subsequently. It is also possible that serum added to sCD4 prebound onto immobilized OKT4 (Fig. 2b) causes significant stabilization by crosslinking of the sCD4-OKT4 complex during subsequent washing steps. This would increase the operational avidity of sCD4 for gp 120. This cannot, however, be the only factor operative when CD4+ sera or MAbs increase the binding of sCD4 to gpl20; thus human sera QS-43, QS-53, and QS-123 and MAb L83 all increased the binding of sCD4 to OKT4, but did not increase binding of gpl20 to sCD4 when they were added to sCD4 after its capture by OKT4. Furthermore, MAbs Q425 and Q428 also increased the binding of recombinant gpl20 to CD4+ cells, whereas a panel of other CD4 MAbs against the third and fourth domains of CD4 had no such effect (D. Healey and P. Beverley, personal communication). Binding CD4+ sera or MAbs to the third and fourth domains of sCD4 probably increases allosterically the affinity of the V1 domain of sCD4 for gpl20, although crosslinking effects may also contribute to the observed ELISA data.

MOORE ET AL. It has been shown previously that blockage of HIV-2 infection of CD4+ cells in vitro requires some 30-fold more sCD4 than needed to inhibit HIV-1 infection of the same cells.6 We have also found that HIV-2 gpl20 has a 25-fold lower affinity than HIV-1 gpl20 for sCD4.18 On the assumption that the two observations were related, we tested whether CD4 autoantibodies and MAbs to the third and fourth domains that increased the binding of sCD4 to gpl20 in ELISA could reduce the concentration of sCD4 required for HIV neutralization, and found that the correlation held. This suggests that the neutralization potency of sCD4 is thus proportional to its affinity for (virion) gpl20, although crosslinking of sCD4 into immune complexes may also contribute to the observed effect. Note that CD4 autoantibodies do not bind to cellular CD4, and so cannot modulate the affinity of the cellular CD4-virion interaction. CD4 autoantibodies and some MAbs to the third and fourth domains of CD4 can therefore increase the binding of sCD4 to gpl20. The binding sites on sCD4 for CD4 autoantibodies that mediate this effect are not known, but the epitopes for the MAbs are currently being delineated more precisely. Knowledge of the regions of sCD4 that indirectly influence its interaction with gpl20 may assist in the design of more potent derivatives of sCD4; for example site-directed mutagenesis of sCD4 regions widely separated in the primary sequence from the gpl20 binding site might improve the therapeutic action of sCD4 in vivo.

ACKNOWLEDGMENTS We are very grateful to Robin Weiss for facilities, advice, and

support. Aine McKnight is thanked for her assistance. We thank

Harvey Holmes of the ADP Resources Programme for several

reagents; Ray Sweet for sCD4; Don Healey and Peter Beverley for MAbs Q425 and Q428 and permission to quote unpublished data; David Buck and Pila Estess for MAbs L83, LI20, and LI88; P. Reiber and G. Reithmuller for MAb MT429; Eddie Follett, Jon Weber, and Clive Loveday for HIV+ sera; Mark Marsh for CBL-JA serum. This work was funded by grants from

the AIDS Directed Programme of the U.K. Medical Research Council to J.P. Moore, to R.A. Weiss, and P.R. Clapham, and to Q.J. Sattentau and M.W. Adler. Additional support was provided by the Cancer Research Campaign and by the European Commission for AIDS Research (EFAR).

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2. Klatzman D, Champagne E, Chamaret S, Gruest J, Guetard D, Hercend T, Gluckman J-C, and Montagnier L: T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature

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Enhancement of soluble CD4-mediated HIV neutralization and gp 120 binding by CD4 autoantibodies and monoclonal antibodies.

We have identified 6 sera containing autoantibodies to CD4 in 174 human immunodeficiency virus-type (HIV-1) positive sera tested in an antigen-capture...
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