Eur. J. Irnrnunol. 1991. 21: 743-751

Anti-CD4 antibodies during HIV infection

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Jean-Philippe Corre., Michkle Fevrier., Sophie Chamaret., Jacques Thkze. and Moncef Zouali.

Anti-idiotypic antibodies to human anti-gpm antibodies bind recombinant and cellular human CD4*

Unite d’lmmunogbnbtique Cellulaire. and Unit6 d’oncologie V i d e A , Institut Pasteur, Paris

The presence of anti-CD4 antibodies in sera of human immunodeficiency virus (HIV)-seropositive individuals has been recently documented, but its origin remains unknown.To test the hypothesis that anti-idiotypic antibodies to gp120, the HIV envelope glycoprotein with high affinity for CD4, mimic the configuration of gp120 and bind CD4, we performed two sets of experiments. First, we tested the possibility that anti-CD4 antibodies present in sera of a proportion of HIV-positive individuals exhibit variable region complementarity to autologous anti-gpl20 antibodies. We show here that affinity-purified human anti-gpl60 antibodies recognize specifically autologous affinity-purified anti-CD4 antibodies. We also demonstrate that antibodies to CD4 competitively inhibit anti-gpl60 autologous antibodies binding to gpl6O.This implies that at least some anti-CD4 antibodies are directed towards idiotypic motifs located on anti-gpl20 antibodies and that they may result from an anti-idiotypic response to anti-gp120 antibodies. In a second set of experiments, we examined the effect of anti-idiotypic immunization of experimental animals against human anti-gpl20 antibodies. We found that anti-idiotypic antibodies produced in a rabbit immunized against affinity-purified human anti-gpl20 antibodies specifically recognize recombinant and cellular human CD4, and that this interaction is competitively inhibited by soluble CD4.The data support the concept of idiotypic mimicry whereby anti-idiotypic antibodies produced against anti-gpl20 antibodies recognize CD4, the cellular receptor of HIV.

1 Introduction The CD4 differentiation molecule is a 58-kDa glycoprotein expressed on the surface of a functionally distinct population of Tcells and of cells of some other lineages. It plays a critical role in the maturation of Tcells and the activation of mature T cells, and this function is likely to rely on interaction of certain domains of the CD4 with nonpolymorphic determinants of MHC class I1 molecules. Recent data indicate that this recognition transduces an intracellular signal through the thyrosine kinase p56Ick.In addition to its role in transmembrane signaling, the CD4 molecule, in concert with other receptors, participates in shaping the T cell repertoire during thymic development (reviewed in [l]). CD4 not only plays a pivotal role in the process of normal immune recognition, but also serves as a cell surface receptor for HIV. This tropism lies in the fact that CD4 interacts with the 120-kDa component of the viral envelope glycoprotein gp120 with high affinity (Kd = 10-9M) (reviewed in [2]). This binding is thought to induce the fusion between the plasma membrane of CD4+ cells and the virus envelope. This will result in entry of HIV reverse transcriptase and RNA into the cytoplasm of the host cell.

By a similar process, interaction between gp120 of infected cells and CD4 of non-infected cells will lead to cell fusion and formation of syncytia [3, 41. There have been recent reports of the presence of antibodies to CD4 in sera of HIV-1-infected individuals [5-71. The origin of this humoral response to CD4 and its effects on viral infectivity of CD4+ cells and on immune-mediated T cell cytotoxicity have not been defined. This study was designed to address several aspects of the origin and the pathological significance of anti-CD4 antibodies. We wished to confirm the presence of antibodies to CD4 in HIV-1-infected individuals and to determine their prevalence in HIV-2-seropositive subjects. We also tested the possibility that anti-idiotypic antibodies to the gp120 molecule may mimic the configuration of gp120 and bind to CD4. In addition, we examined the effect of anti-CD4 antibodies on HIV in vitro infection and on immunemediated cytotoxicity of human CD4+ lymphocytes.

2 Materials and methods 2.1 Subjects

Serum samples were obtained from 93 HIV-1- and 10 HIV-Zinfected individuals. These seropositive subjects were either asymptomatic, or exhibited clinical manifesta[I 88521 tions in the form of persistent generalized lymphadeno* This work was supported by a research grant from the “Agence pathy, AIDS-related complex, or AIDS. Sera were heatinactivated for 1 h at 56°C and then stored at - 20°C. One Nationale de Recherche sur le SIDA”, France to M.Z. hundred control sera were drawn from normal blood donors Correspondence: Moncef Zouali, Institut Pasteur, Immunogknd- through the “Fondation Nationale de Transfusion Sanguine” (Paris). tique Cellulaire, 28, rue du Dr. Roux, F-75015 Pans, France 0 V C H Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

0014-2980/91/0303-0743$3.SO + .25/0

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J.-P Corre, M . Fkvrier. S. Chamaret et al.

2.2 Recombinant proteins and mAb

2.6 Cell lines

Human soluble CD4 was kindly donated by Dr. J. Mous (E Hoffmann-La Roche AG, Basel, Switzerland) and Drs. S. Szmelcman and M. Hofnung (Institut Pasteur, Paris). Both of these molecules consist of the first amino-terminal 177 amino acids, including the V1 and V2 domains of CD4. Samples of gp160 of HIV-1 isolate B R U were obtained from Drs. M. Girard and M. Kaczorek (Pasteur Vaccins,Val de Reuil, France). The PB-1 antigen, encompassing amino acids 295-474 of gp120 of HIV-1 isolate IIIB, was provided by Dr. S. Putney (Repligen Corporation, Cambridge, MA). Anti-CD3 (OKT3) and anti-CD4 (OKT4a) mAb were from Ortho Diagnostic Systems, Inc. (Westwood, MA.). AntiCD4 mAb IOT4-B14 was purchased from Immunotech S.A. (Marseille, France). mAb 13B8, was a gift of Drs. D. Olive and C. Mawas (Centre d’Immunologie de MarseilleLuminy, France).

Two human CD4+ Tcell lines (CEM and HPB-ALL) were used. They were kindly provided by Drs. B. Dupont and S. Chouaeb (Sloan Kettering Cancer Institute, New York, NY). The CD4- human acute lymphoblastic leukemia cell lines HSB-2 and C-10 were obtained from Drs. B. Ardman (Tufts University, Boston, MA) and R. Gallo (National Cancer Institute, Bethesda, MD), and from Dr. P A. Cazenave (Institut Pasteur, Paris), respectively.

2.3 Radioimmunoprecipitation assays for detection of antibodies to HIV-1 Radioimmunoprecipitation assays (RIPA) were carried out using extracts from labeled HIV-l-infected cells as described [8]. Briefly, proteins were metabolically labeled by culturing HIV-l-infected cells for 16 h at 37°C in MEM culture medium devoid of L-methionine and serum, but supplemented with 200 pCi/ml (= 7400 MBq/ml) ~-[”S]methionine (Amersham, Les Ulis, France). Radiolabeled cells and viral lysates were diluted in RIPA buffer, incubated with dilutions of test sera or purified antibodies, and processed as described previously [8]. 2.4 ELISA for antibody activity against CD4 Microtiter plates (Nunc, Roskilde, Denmark) were coated with soluble recombinant CD4 at 1 pg/ml in 0.1 M sodium borate buffer, pH 8.4. Dilutions of serum or purified Ig to be tested were revealed with a 2 x dilution of an alkaline phosphatase-labeled, Fc-specific goat anti-human IgG or IgM F(ab’h fragments (Sigma Chemical Co., St. Louis, MO). All dilutions were made in PBS containing 1% BSA (PBS-BSA) and 0.1% Tween 20, and plates were washed three times between each step with PBS containing 0.1% Tween-20 (PBS-Tween 20). At each step, incubation was carried out at 37°C for 2 h. After the last washing, 100 pl of the alkaline phosphatase substrate (1 mg/ml p-nitrophenyl phosphate dissolved in 0.1 M sodium carbonate buffer, pH 9.2 containing 1 mM MgC12) were added and the plates incubated for 30 min at 37°C. Absorbance was recorded at 405 nm with a Dynatech Multiscan.

2.7 Immunofluorescence Detection of antibody activity to cellular CD4 was also determined by immunofluorescence FCM using two CD4+ human T cell lines (CEM and HPB-ALL). For each analysis, 5 x los cells were exposed to various dilutions of test sera or test antibodies, and then stained with an optimal concentration of FITC-conjugated F(ab’)z fragments of goat antibodies to human Ig (Ortho Diagnostic Systems, Inc., Westwood, MA). Each incubation was carried out in PBS-BSA containing 0.05% NaN3 for 30 min at 4 ° C in V-bottom microtiter plates, and then followed by three washings in the same buffer. Stained cells ( l v ) were fixed with 1% paraformaldehyde and run on a FACS analyzer (Becton Dickinson, Mountainview, CA) and data were analyzed with the use of a Consort 30 software. 2.8 lmmunoaffinity isolation of antibodies Three affinitycolumns were obtained by immobilizingCD4 (700 pg), PB-1 (1 mg), and gp160 (1.1 mg) on CNBractivated Sepharose-4B (2 ml) according to the procedure recommended by the manufacturer (Pharmacia Fine Chemicals, Sweden). The Ig fractions of the seropositive sera were passed over the columns and, after extensive washings with PBS, bound materials were eluted with 3 M KSCN buffer, and immediately dialyzed against PBS. 2.9 Antibody labeling Affinity-purified antibody (10 mg/ml) was dialyzed against 0.15 M NaCl buffer. Labeling was performed by mixing 500 p1 of dialyzed antibody, 50 p1 of 1 M NaHCOB buffer and 50 pl of D-biotin-N-hydroxy-succinimidester (Boehringer Mannheim, Mannheim, FRG; 75 mg/ml) dissolved in dimethylformamide. After a l-h incubation at 22 “C, the labeled antibody was extensively dialyzed and immediately stored at - 20°C. 2.10 Generation of anti-Id antibodies

2.5 Data analysis The mean absorbance at 405 nm of duplicate assays were calculated, and values obtained with serum dilutions of 100 seronegative healthy individuals were used for determining a cut off for distinguishing “positives” from “negatives”. A value of 3 S D above the mean was chosen as a cut-off value. A serum was considered positive when the mean absorbance of duplicate measurements was > 3 SD, and negative if the mean absorbance was lower.

Anti-Id antisera to affinity-purified human anti-PB-1 antibodies (IgG) were raised in rabbits by i.m. injections in adjuvant at 3-week intervals, first in CFA and then in IFA. For the first injection, 50 pg of antibody was used.This was increased to 100 pg for the six injections which followed, and blood samples were collected at regular intervals. Rabbit antisera were absorbed extensively on affinity columns of Ig isolated from a pool of human sera which lacked detectable anti-PB-1 antibody activity as described [91.

Anti-CD4 antibodies during HIV infection

Eur. J. Immunol. 1991. 21: 743-751

2.11 Assay for antibody-mediated cytotoxicity (ADCC), for C-dependent lysis and for virus neutralization

The ADCC was determined as described previously [ 101 using CD4+ CEM cells and peripheral blood mononuclear cells from normal healthy donors as targets and effector cells, respectively. In brief, 5 x 106 target cells resuspended in serum-free medium were labeled with 200 pCi of 51Cr (Na2 51Cr04) for 1 h at 37°C. After washing, 104 labeled CEM cells were incubated in round-bottom 96-well plates with 50 pI of test sera or purified antibodies for 30 min at 37°C. Effector cells were isolated on a Ficoll-Hypaque gradient and added at an E/T ratio of 50/1. 51Crrelease was measured after incubation in a 6.5% C 0 2 incubator for 4 h. C-dependent lysis was determined by incubating various amounts of purified anti-CD4 antibodies to be tested with 104 51Cr-labeledCD4+ CEM cells. After 1 h at 2 2 ° C an appropriate dilution of C was added and specific lysis was determined in a 4-h 51Cr-release assay. The virus neutralization assay was performed as follows. Serial dilutions of test antibodies were incubated with 10" CEM cells (clone 13) for 1 h at 37 "C in round-bottom microtiter plates.Virus SN (equivalent of 105 cpm reverse transcriptase activity) was added and the cells were cultured for 13 days in a 5% C 0 2 incubator. Viral infectivity was determined on days 6 and 13 by measurement of reverse transcriptase production and cell viability using the tetrazolium salt

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Figure 2. Binding of murine anti-CD4 mAb to human recombinant CD4. Various dilutions of the mAb were incubated with

CDCcoated wells and, after washing, bound antibodies were revealed by an alkaline-phosphatase-labeledgoat anti-mouse Ig F(ab)2 fragments. (A-A) Anti-CD4 mAb 13B8; (m-m) anti-CD4 mAb IOT4-B12; (0-0) anti-CD3 mAb OKT3.

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3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetr~olium bromide assay as described previously [11J .

3 Results 3.1 Antibody activity to recombinant CD4 in HIV+ sera In initial screenings, 93 HIV-1+ and 10 HIV-2+ sera diluted 1/50 and 1/500 were tested by ELISA for binding to soluble CD4. Using a threshold value of 3 S D of the average absorbance of 100 seronegative sera, 7 HIV-l+ sera and one

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Figure 1. Binding of human HIV+ sera to human recombinant CD4. Various dilutions of human sera were incubated with CDCcoated wells and, after washing, bound antibodies were revealed by an alkaline phosphatase-labeled goat anti-human Ig F(ab')z fragments. Represented are the binding curves of five HIV-l+ sera [serum 2917 (A-A); serum 2112 (m-m); serum 2923 (0-0);serum 2965 (A-A); and serum 3038 (0-0)] and one HIV-2+ serum (0-0).Also shown are the mean absorbances of 100 anti-CD4-negative control sera (x-x).

Figure 3. Isotypic patterns of human anti-CD4 antibodies. The experimental procedure was as described in legend to Fig. 1 . except that antibody binding was revealed by either an anti-human IgG or an anti-human IgM enzyme-labeledreagent. Shown are the data obtained with two HIV+, anti-CD4+ sera, and one HIV+, anti-CD4- serum. (m-m) HIV-I+ serum (2923) binding to CD4 revealed by secondary antibody to human IgG. (13-13) Serum 2923 binding to CD4 was revealed by a secondary antibody to human IgM. (A-A) H1V-F serum (41) binding to CD4 revealed by a secondary antibody to human IgG. (A-A) Serum no. 41 binding to CD4 was revealed by a secondary antibody to human IgM. (0-0)An anti-CD4- serum was used together with a secondary antibody to human IgG. (0-0)The anti-CD4- serum was used together with a secondary antibody to human IgM.

746

Eur. J. Immunol. 1991. 21: 743-751

J.-€! Corre, M. Fevrier, S. Chamaret et al.

HIV-2+ serum exhibited anti-CD4 activity. The sera that scored positive in the initial screening were then retested by ELISA at various dilutions ranging from 1/50 to 1/51200. Representative binding curves of these positive sera are shown in Fig. 1. For comparison the binding curves of two murine anti-CD4 mAb are represented in Fig. 2. We next determined the isotype of these anti-CD4 antibodies by an ELISA using alkaline phosphatase-labeled anti-human IgG and anti-human IgM F(ab’)zfragments.Wefound that each serum exhibited a unique isotypic pattern : in some sera IgG antibodies predominated, and in others anti-CD4 antibodies were of the IgM and IgG isotypes (Fig. 3). 3.2 Binding of human anti-CD4 antibodies to CD4+ cells

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To ascertain that antibodies recognizing soluble CD4 could bind native cellular CD4, serum samples which scored positive in the anti-CD4 ELISA were tested for binding to CD4+ cells by FCM. To achieve this, we used subclones of two CD4+ cell lines (CEM and HPB-ALL) that expressed high levels of cell surface CD4 as tested by fluorescence using the murine OKT4a anti-CD4 mAb. In contrast to anti-CD4- sera which did not show any binding to CD4+ clones, the 8 anti-CD4+ sera diluted 1/50 and 1/500 recognized these cells (Fig. 4). When twofold dilutions (1/100 to 1/3200) of these sera were tested, there was a dilution-dependent variation of the fluorescence signal. In contrast,when the human CD4- cell lines HSB-2 and C.10

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Figure 5. Binding of affinity-purified anti-gpl60 and anti-CD4 antibodies isolated from the serum of a single individual (PLU). Various dilutions of the affinity-isolated antibodies were incubated with CD4- or gpl60-coated wells and bound antibodies were revealed by an alkaline phosphatase-labeled anti-human Ig reagent. In (A) gp160 was the test antigen immobilized on the solid phase: (B-B) affinity-purified anti-gpl60 antibodies, (0-0)control human Ig. In (B) CD4 was the test antigen immobilized on the solid phase: (0-0) affinity-purified anti-CD4 antibodies, (0-0) control human Ig.

were incubated with the anti-CD4+ sera, no fluorescence signal was detectable. We therefore conclude that 7.7% of HIV+ sera bind soluble and cell surface human CD4.

Green Fluorescence Intensity

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Figure 4. Binding of a human HIV-I+ serum to CD4+ CEM cells by FCM. CEM cells were incubated with a 1/500 dilution (broken line) or a 1/50 dilution (solid line) of an anti-CD4+ serum or a 1/50 dilution of an anti-CD4- human serum (dotted line). After washing the cells were incubated with FITC-conjugated goat anti-human Ig F(ab’)z fragment as a secondary antibody. By comparison,when the human CD4- cell lines HSB-2 and C-10 were incubated with the anti-CD4+ sera, no fluorescence signal was detectable.

We attempted to correlate the presence of antibodies to CD4 with clinical manifestations, antibodies to HIV core antigen, and CD4+ Tcell counts. Consistent with previous observations [5-71, this analysis failed to reveal any correlation between the presence of anti-CD4 antibodies and the changing parameters of disease progression. To gain insight into the origin of anti-CD4 antibody production,we hypothesized that, following HIV infection, anti-gpl20 and anti-anti-gpl20 (anti-Id) antibodies are generated, and that some of these anti-Id may be directed to CD4.Two sets of experiments were performed to test this hypothesis. 3.3 ldiotypic interactions between human anti-CD4 antibodies and autologous anti-HIV antibodies

If anti-CD4 antibodies present in the sera of certain HIV-infected individuals were in fact the result of Id-anti-Id interactions, their binding ability should be idiotypically connected to that of autologous anti-gpl20 antibodies. To

Eur. J. Immunol. 1991. 21: 743-751

Anti-CD4 antibodies during HIV infection

747

demonstrate this, an anti-CD4+ human serum termed PLU was selected for further study. Antibodies to gp160 were affinity isolated from this serum, and then labeled wilth biotin. Autologous antibodies to CD4 were also purified on an affinity column of recombinant human CD4 (Figs. $ 6 ) . To make certain that Ig without anti-gpl60 or anti-CD4 activity were not eluted from these affinity columns, a pool of biotin-labeled normal human Ig were passed over these columns. No detectable proteins could be eluted with 3 M 1 KSCN, as assayed by spectrophotometric analysis, by the l o o 0 4 0 0 0 S O 00 Bradford protein assay, or by the avidin-alkaline phosphaLobolod Anli-gpl60 Anllbody (ng/ml) tase detection system. In other experiments, a HIV+, anti-CD4+ serum was passed over a Sepharose column to which no ligand was coupled. After washing with PBS, no Figure 7. Binding of labeled anti-gpl60 antibodies to autologous detectable proteins were found in the eluate.To rule out the anti-CD4 antibodies. Serial dilutions of biotin-labeled anti-gpl60 possibility that, as a result of column leakage, the affinity- antibodies were incubated with wells coated with affinity-purified autologous anti-CD4 antibody (W-W), or the murine mAb IOT4 or purified anti-CD4 and anti-gpl60 antibodies were contam- with control human Ig (0-0).Bound antibodies were revealed by inated with CD4 and gp160, respectively, the antibody adding an alkaline phosphatase-labeled streptavidine reagent. preparationswere submitted to polyacrylamide gel electrophoresis. This analysis did not reveal the presence of CD4 and gp160 proteins. column and eluted with KSCN were tested for binding to Even though the recombinant CD4 used to prepare the CD4 immobilized on the soIid phase. Under these condiaffinity column was in a highly purified form,we performed tions, the material tested had no binding capacity to the additional experiments to exclude the possibillity that CD4 CD4 preparation, as compared to normal human Ig that was contamined by bacterial components, and that the have not been passed over the CD4 column. affinity-purified antibody eluted from this column contains subsets directed against the contaminants. Biotin-labeled Assays for putative Id interactions were then performed normal human Ig that have been passed over the CD4 with affinity-purified anti-gpl60 and anti-CD4 antibodies isolated from the serum of the same subject. As can be seen in Fig. 7, labeled anti-gpl60 antibodies were able to bind to autologous anti-CD4 antibodies immobilized on microtiter wells. In contrast, they failed to recognize a pool of normal human Ig and anti-CD4 antibodies from ZAN human serum.This latter finding is compatible with related studies showing that anti-gpl20 antibodies from unrelated seropositive individuals do not share common idiotopes (Fdvrier M. et al., manuscript in preparation). To see whether the activity of anti-gpl60 antibodies is specific for the autologous anti-CD4 antibody, or whether it is also directed against other anti-CD4 antibodies, we tested the biotin-labeled anti-gpl60 human antibody for binding to the murine anti-CD4 IOT4 mAb.We found that the human FLP anti-gpl60 antibody did not recognize this xenogeneic anti-CD4 antibody. Y

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Green Fluorescence Intensity Figure 6. Binding of anti-CD4 antibodies isolated from human serum PLU to CD4+ CEM cells. Control human Ig (dotted line) and PLU anti-CD4 antibodies (solid line) were diluted at a concentrationof 10 pg/ml and incubated with the CD4- HSB-2 cell line (A) or with the CD4+ CEM cell1 line (B). After washing, the cells were incubated with FITC-conjugated goat anti-human Ig F(ab’)z fragments as a secondary antibody.

That the affinity-purified anti-CD4 and anti-gpl60 antibody preparations were contamined with CD4 and gp160, respectively, that may have leaked from the affinity columns was ruled out as follows. First, when a pool of normal human was passed over the CD4 column, eluted with KSCN and then used as the coating antigen in ELISA, the biotin-labeled anti-gpl60 antibody exhibited no binding activity towards this immobilized material. Second, when the normal human Ig were passed over the gp160 column, biotin-labeled, and then tested for binding to the solidphase affinity-purified human anti-CD4 antibody, no binding was detectable.

To demonstrate more rigorously that anti-CD4 antibodies interact with autologous idiotypic determinants of antigp160 antibodies, we tested their inhibitory potential on gp160-anti-gp160 recognition. Preincubation of labeled anti-gpl60 antibodies with autologous affinity-isolated anti-CD4 antibodies decreased their gpl60-binding capac-

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3.4 Experimental immunization against human anti-gplU) antibodies generates antibodies to human CD4

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The serum of another HIV-1+ individual with no clinical manifestation of disease was selected. Anti-gpl20 antibodies were purified from the serum termed ZAN on a column of PB1 immobilized on Sepharose4B beads. PB1 is a 180-amino acids recombinant protein encoded by the gene corresponding to the restriction fragment Bg 11-Pvu I1 of gp120 of HIV-1 isolate IIIB and encompassing positions 295 through 474 [12] of the protein.This molecule was used as a substitute of gp120 because large quantities of gp120 were not available at the time we performed these experiments. Affinity-purified ZAN anti-PB1 antibodies had the following properties : (a) in ELISA they bound PB1 and recombinant gp160, (b) their binding to gp120 could be detected by Western blotting, (c) they immunoprecipitated metabolically labeled gp120 and gp160 in a RIPA system, and (d) they recognized HIV-1-infected cells, but not control cells, as detected by indirect immunofluorescence. This anti-PB1 antibody preparation will be referred to as ZAN anti-gpl20 antibodies or Abl.

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Eur. J. Immunol. 1991. 21: 743-751

J.-I! Corre, M. Fkvrier, S. Chamaret et al.

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Figure 8. Inhibition of labeled anti-gpl60 antibody binding to gp160 by gp160 and by autologous anti-CD4 antibodies. A concentration of biotin-labeled anti-gpl60 antibodies that achieved 60% of maximal binding to solid-phase gpl60 was incubated with various dilutions of competitors.The mixtures were then transferred to gpl60-coated wells and bound antibodies were revealed by adding an alkaline phosphatase-labeled streptavidine reagent. In ( A ) recombinant gpl60 (M-.) and OVA (0-0)were used as competitors. In (B) affinity-purified anti-CD4 antibodies (0-O), purified control normal Ig and control normal Ig that have been passed over the CD4 affinity-column and eluted with KSCN (0-0) were used as competitors.

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ity (Fig. 8). When the pool of normal human Ig that had been passed over the CD4 column and eluted with KSCN was used as competitor of gpl60-affinity-purified antigp160 interaction, no inhibition was seen. This finding excludes the possibility that the affinity-purified anti-CD4 preparation was contaminated with CD4. It is of note that at similar molar concentrations, recombinant gp160 was more effective than anti-CD4 antibodies in inhibiting gp160-anti-gp160 interaction. This is not unexpected; indeed, anti-gpl60 antibodies consist of antibodies to gp120 and to gp41, but anti-CD4 (anti-anti-gpl20) antibodies are theoretically able to interfere with only antigp120 binding to gp160. Presumably, anti-CD4 antibodies can only block a subset of anti-gpl60 antibodies. These experiments show that, at least in this HIV-infected individual, anti-CD4 antibodies are directed towards Id motifs located on anti-gpl60 antibodies, and that they may result from an anti-Id response to anti-gpl20. To test this assumption, we examined the effect of anti-Id immunization of experimental animals against human anti-gpl20 antibodies.

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Figure 9. Binding of anti-ZAN anti-Id antibodies to recombinant human CD4. Rabbit B was immunized against affinity-purified human Z A N anti-gpl20 antibody and the immune serum was rendered Id specific by passing several times over a column of normal human Ig.The eluate of this column, which contains rabbit antibodies to the Fc portion of human Ig (m-.), and the flow-through, which contains anti-Id antibodies to ZAN (0-0). were tested for binding to recombinant CD4 and to normal human Ig by ELISA. In (A) normal human Ig were used as the solid-phase test antigen; in (B) recombinant human CD4 was used as the solid-phase test antigen.

Eur. J. Immunol. 1991. 21: 743-751

Anti-CD4 antibodies during HIV infection

Rabbits were immunized against Abl, and rabbit B which developed a high-titer anti-Id response was selected for further study. Rabbit B immunoserum was rendered Id specific by extensive absorption on a column of a pool of normal human Ig, and then tested for binding to soluble CD4 by ELISA. Fig. 9 shows that the anti-ZAN anti-Id reagent recognizes CD4 but not normal human Ig. In contrast, the material eluted from the normal human Ig column binds to normal Ig, but not to soluble CD4. This result demonstrates that only the anti-ZAN anti-Id fraction of the immune rabbit serum contains anti-CD4 activity and that antibodies to the constant portion of human Ig are devoid of anti-CD4 activity. Antibodies to CD4 were then isolated by immunoaffinity on a CD4 column (Fig. lo), and their binding to soluble CD4 was shown to be inhibitable by CD4, but not by irrelevant proteins (Fig. 11).

To provide further evidence for the binding affinity of anti-ZAN anti-Id antibodies to CD4. we tested their

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Green Fluorescence Intensity Figure 12. Binding of rabbit anti-Id antibodies to CD4+ CEM cells by FCM. CEM cells were incubated with rabbit anti-Id antibodies to ZAN anti-gpl20 antibodies (solid line) or with control normal rabbit Ig (dotted line). Both Ig preparations were tested at a concentration of 10 pg/ml. After washing, the cells were incubated with FITC-conjugated goat anti-rabbit Ig as a secondary antibody. By comparison, when the human CD4- cell lines HSB-2 and C-10 were incubated with the anti-Id antibodies, no fluorescence signal was seen.

activity on cell surface CD4 by FCM. In contrast to normal rabbit Ig, anti-ZAN Ig bound human CD4+ cells (Fig. 12) and this binding varied in a dose-dependent manner. In contrast, when the human CD4- cell lines HSB-2 and C-10 were incubated with the anti-idiotypic antibodies, no fluorescence signal was seen. These experiments show that experimental immunization against human anti-gpl20 antibodies elicits anti-idiotypic antibodies able to recognize CD4, the natural cellular receptor of HIV.

Antibody Concentration (yglmi)

Figure 10. Binding of affinity-purified rabbit anti-CD4 antibodies to recombinant human CD4. Anti-CD4 antibodies were affinity purified from the serum of rabbit B immunized against ZAN human anti-gpl20 antibodies and tested for binding to recombiby ELISA. nant CD4 (m-m) and CD8 (0-0)

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Among the numerous immunological abnormalities that have been described in the course of HIV infection (reviewed in [13,14]), the presence of antibodies to CD4 in the sera of seropositive individuals [5-7, 151 has attracted much attention, mainly because the CD4 molecule is the major cellular receptor of the HIV virus. This study was designed to gain insight into the origin of these CD4binding antibodies, and the data suggest that, at least in some individuals, an anti-idiotypic response to gp120 may give rise to antibodies to the CD4 receptor.

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To gain insight into the biological significance of ant i-CD4 antibodies, we examined their effect on HIV infection and we tested their potential to exert C dependent cytotoxicity and ADCC. Under the experimental conditions we have used, no significant activity of anti-CD4 antibodies could be found.

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Figure 11. Inhibition of rabbit anti-CD4 antibodies binding to CD4. One hundred microliters of serial dilutions of competitors was preincubatedwith an equal volume of anti-CD4 antibodies at a concentration that achieved 60% of maximal binding to CD4.The mixtures were then transferred to CDCcoated wells and bound antibodies were revealed by adding an alkaline phosphataserecombinant human CD4; labeled anti-rabbit Ig reagent. (m-.) (0-0) OVA.

Earlier reports [5-7, 151 indicated that the prevalence of anti-CD4 antibodies in HIV-1-infected individuals varies between 5.2% [15] and 12.6% [6]. The figure of 7.7% we have observed is therefore consistent with these previous reports. We additionally made the observation that HIV2-infected subjects may develop antibodies to CD4. This finding is not unexpected because both HIV-1 and HIV-2 viruses bind to highly conserved regions of the CD4 receptor [16].

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Since antibodies to CD4 have been detected in approxi- possible that regulatory mechanisms of immune homeostamately 10% of seropositive subjects ([6]; this report) their sis may down-regulate activation of these autoreactive significance with regard to pathogenesis might be ques- B cells and prevent anti-CD4 antibody production. Third, tioned. However, the assay systems that we and others the prevalence of actual anti-CD4 production in vivo may [5-71 have used to detect anti-CD4 activity were not be > lo%, but, as discussed above, the assay systems used appropriate to detect all putative immune responses to result in underestimation of the frequency of the anti-CD4 CD4. First, if antibodies to CD4 were trapped in circulating response. CD4-anti-CD4 immune complexes, they would have been undetectable by these assays. Second, anti-CD4 antibodies Our experimental production of anti-Id antibodies to HIV of the IgA class were not searched for.Third, antibodies to with affinity for the viral receptor recalls earlier data CD4 bound to the surface of circulating CD4+ cells would described for the polyomavirus [22] and reovirus type 3 have been missed in the assays used. Therefore, anti-CD4 [23]. In this latter system, anti-Id mimic the reovirus by antibodies present in the positive sera were probably attaching to specific cellular receptors, compete with the underestimated in this and previous reports [5-71. Finally, it virus for attachment to these receptors, and some of them is also conceivable that cellular anti-CD4 elements are elicit both humoral and cell-mediated immune responses generated in the course of HIV infection. Other studies will ~ 3 1 . be required to address these issues. Related to our findings are reports of anti-Id antibodies to The origin of anti-CD4 antibody production during HIV CD4. Experimentally, immunization of mice with a murine infection has not been elucidated. Four potential mecha- anti-CD4 mAb, Leu3A, elicited Ab2 which mimic the CD4 nisms may be entertained. First, polyclonal activation receptor, react with HIV and with HIV-infected cells, bind occurs early in HIV infection [17] and can theoretically to gp120, and weakly cross-neutralize diverse HIV isolates result in anti-CD4 antibody production. Second, destruc- [21,24]. However, since mAb were not derived from these tion of CD4+ lymphocytes in the course of HIV infection Ab2-producing animals, it remains uncertain whether the may lead to an immune response to CD4. Third, binding of neutralization activity results from Ab2 mimicking the CD4 gp120 to CD4 may alter CD4 motifs that become accessible receptor and binding to gp120, or from anti-Ab2mimicking and/or immunogenic to the immune system. However, gp120 and blocking CD4. It is noteworthy that further there are no data to support the idea that exposure of novel mapping studies [25] have revealed that Leu3A antibody CD4 epitopes results from gp120-CD4 interaction. Fourth, does not precisely mimic gp120 binding to CD4.This could anti-Id to the nominal antigen gp120 of HIV represent explain the weak neutralization response obtained. internal images of gp120 and, therefore, bear complementarity to, and recognize, the cellular receptor CD4. Our Along the same line, other investigators addressed the issue findings are consistent with this latter possibility, but they of whether sera from HIV-infected individuals contain do not formally exclude the other mechanisms of anti-CD4 antibodies with dual capacity to bear the structural conforproduction. This conclusion is based on two lines of mation of CD4 and to display specific affinity towards the evidence. First, we have shown that autologous anti-gpl20 gp120 protein of HIV [26].They reported the occurrence of and anti-CD4 antibodies fractionated on affinity columns anti-idiotypes to an anti-CD4 mAb,T4-2, at a frequency of exhibit binding complementarity.Second, we demonstrated 5.2%.Their data also suggested that the presence of Abz to that experimental immunization of animals against anti- CD4 may be of benefit for the patient. gp120 antibodies elicits anti-Id antibodies with affinity for In a recent report, it was found that a proportion of soluble and cellular CD4. polyclonal human Ig and human myeloma proteins bind Initially, Jerne et al. [18] proposed a formal distinction CD4 and that this binding is probably mediated by the Fd between two sets of anti-Id (Ab2) antibodies. The Ab2a region (V, plus CHI) of the Ig molecule [27]. On the recognizes idiotopes located at a distal position from the surface, it may appear that this finding contradicts the paratope of the antibody (Abl), and does not interfere with results of the present work and those of related investigaAbl binding to the nominal epitope. The Ab2P is directed tions [5-71. More likely, two lines of evidence may account towards the paratope of Abl and bears the “internal image” for this apparent discrepancy. First, in the report describing of the antigen. Anti-Id mimicking the structural configura- the binding of normal human Ig to CD4 [27], saturation of tion of the nominal antigen were described in studies of binding, as detected by ELISA, was obtained at 40 pg/ml antigens of various origins, including tumors and pathogens for IgG, IgM, IgD and IgE, and at 80 vg/ml IgA. By such as parasites, bacteria and viruses (reviewed in [19]). comparison, a murine mAb specific for CD4 reached a Several investigators have proposed that anti-gpl20 anti- plateau at 4 pg/ml. In that same report [27], human bodies produced in response to HIV infection might elicit polyclonal Ig tested at 100 pg/ml were found to bind CD4+ anti-Id antibodies that mimic gp120 and bind CD4 [20,21]. CEM cells by FCM. Here, we found that when normal Our findings provide support for this hypothesis. As human Ig were tested at 5 pg/ml by ELISA, and at 10 pg/ml discussed earlier, the incidence of anti-CD4 antibodies in by FCM, no significant binding to CD4 was detectable. seropositive individuals is, however, consistently low Other authors using the same range of Ig concentrations throughout the different studies [5-71. We would like to reached the same conclusions [5-71. It is, therefore, likely offer three non-mutually exclusive explanations. First, an that the anti-CD4 antibodies present in HIV+ individuals MHC-linked genetic restriction of the anti-Id response (this work, [5-71) are of higher affinity than those found could account for this variability and it may be that only among normal human Ig [27]. Second, it is well known that a small proportion of B lymphocytes of normal individuals “high responders” will develop such anti-Id. Second, Ab$ mimicking HIV motifs and reacting with the cellular bind a variety of autoantigens, that Ig purified from normal receptor CD4 behave like autoantibodies. It is therefore human sera exhibit binding specificity for autoantigens, and

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that a proportion of human myeloma proteins express an autoantibody activity of low to moderate affinity [9,28,29]. This binding activity may be directed towards autologous idiotopes [30, 311. Therefore, it may not be too surprising that certain human Ig exhibit an autoantibody activity to self motifs of CD4 [27]. Considerableattention has been paid to the use of anti-Id to manipulating the functional arms of the immune system (reviewed in [32-341. Our data show that production of anti-gpl20 antibodies triggers synthesis of anti-Id antibodies that will bind to CD4. However, the functional activity of these anti-CD4 antibodies remains unknown. It is conceivable that they might contribute to the induction of immune dysfunction in HIV-infected individuals leading to progressive impairment of the immune response. It is also possible that they might play a protective role by competing with viral binding to the CD4 receptor. The seropositive individual PLU with high titers of anti-CD4 antibodies has not developed clinical symptoms. However, we were unable to detect an in virro functional activity of purified PLU anti-CD4 antibodies. It is important to note that the tests we have used are not very sensitive. For example, a concentration of 50 pg/ml of an anti-CD4 mAb is required to inhibit viral infection in our assay. More sensitive tests are therefore required to address this issue. We are grateful to Drs. J. Mous (Hoffman Laroche, Switzerland), M . Hofnung and S. Szmelcman (Institut Pasteur, Paris), and H. Wigzell for provisions of recombinant CD4. We are indebted to Drs. M . Girard and M . Kaczorek (Pasteur Vaccins, Val de Reuil, France) for generous gifts of recombinant gpla). We thank Dr. S. Putney (Repligen Corporation, Cambridge, MA.) for providing us with the PB-I molecule, Y Henin (Oncologie Virale, Institut Pasteur, Paris) for performing inhibition assays, Dr. t? Lambin (Fondation de Transfusion Sanguine, Paris) for the gift of sera and Dr. L . Montagnier (Oncologie Virale, Institut Pasteur, Paris) for critical reading of the manuscript. We also thank Drs. D. Olive and C. Mawas (Centre d’lmmunologiede Marseille-Luminy) for the gift of monoclonal antibodies. We appreciate the expert secretarial assistance of Annick Bas.

Received August 29, 1990; in final revised form November 8, 1990.

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