`Vol. 84, pp. 3891-3895, June 1987
`Medical Sciences
`
`Monoclonal anti-idiotypic antibody mimics the CD4 receptor and
`binds human immunodeficiency virus
`
`(acquired immunodeficiency syndrome/ receptor mimicry /T -lymphocyte surface molecule)
`
`TRAN C. CHANH, GORDON R. DREESMAN, AND RONALD C. KENNEDY*
`Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, TX 78284
`
`Communicated by Alfred Nisonoff, February 20, 1987
`
`A monoclonal anti-idiotypic (anti-Id) anti(cid:173)
`ABSTRACT
`body, HFl.7, was generated against anti-Leu-3a, a mouse
`monoclonal antibody (mAb) specific for the CD4 molecule on
`human helper/inducer T lymphocytes. The anti-Id nature of
`HFl. 7 was demonstrated by the following properties. (i) It
`reacted in a solid-phase immunoassay with anti-Leu-3a and not
`with a panel of irrelevant mouse mAbs. (ii) It partially inhibited
`the binding of anti-Leu-3a to c04+ T cells. (iii) It detected a
`common idiotype present on various anti-CD4 mAbs. Because
`the CD4 molecule represents the receptor site for human
`immunodeficiency virus (HIV), the etiologic viral agent of
`acquired immunodeficiency syndrome, we examined the ability
`of the anti-Id mAb HFl.7 to mimic CD4 and bind HIV. This
`anti-Id mAb reacted with HIV antigens in commercial HIV
`ELISAs and recognized HIV-infected human T cells but not
`uninfected cells when analyzed by flow cytofluorometry. At(cid:173)
`testing further to the HIV specificity, the anti-Id mAb reacted
`with a recombinant gp160 peptide and a molecule of M,
`110,000-120,000 in imm1inoblot analysis of HIV-infected cell
`lysates. The anti-Id mAb also partially neutralized HIV infec(cid:173)
`tion of human T cells in vitro .. These results strongly suggest
`that this anti-Id mAb mimics the CD4 antigenic determinants
`involved in binding to HIV.
`
`Acquired immunodeficiency syndrome (AIDS) is a devastat(cid:173)
`ing disease resulting from infection of many cellular compo(cid:173)
`nents vital for the maintenance of immune homeostasis.
`Human immunodeficiency virus [HIV; also called human
`T-lymphotropic virus type III (HTLV-III), lymphadenopa(cid:173)
`thy-associated virus (LAV), and AIDS-associated retrovirus
`(ARV)], the etiological agent of AIDS, is lymphotropic for
`cells expressing the CD4 molecule. HIV has been shown to
`infe~t not only the helper/inducer subset of T lymphocytes
`but also cells of the monocyte/macrophage lineage (1-4). In
`vitro infection by HIV can be effectively blocked by mono(cid:173)
`clonal antibodies (mAbs), such as anti-Leu-3a and OKT4A,
`directed against the CD4 target molecule (4-6). It has been
`shown recently (7) that HIV binds to the CD4 molecule via
`an envelope glycoprotein of M, 110,000. These results imply
`that tbe CD4 antigenic determinants recognized by anti-Leu·
`3a and OKT4A either represent the site of attachment of HIV
`or are closely associated with it. Based on Jeme's idiotype
`network hypothesis (8), anti-idiotype (anti-Id, or Ab-2)
`against anti-Leu-3a or OKT4A (Ab-1) bearing the internal
`image should mimic the antigen (CD4) and bind to HIV
`envelope glycoprotein. This interaction in tum may inhibit
`the binding of HIV to CD4 on target cells and therefore could
`lead to viral inactivation.
`A monoclonal anti-Id antibody, termed HFl.7, was gen(cid:173)
`erated against mAb anti-Leu-3a. HFl.7 exhibited the follow(cid:173)
`ing properties. (i) It reacted in solid-phase enzyme-linked
`
`The publication costs ofthis article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`immunosorbent assay (ELISA) with anti-Leu-3a and not with
`a panel of irrelevant mouse mAbs. (ii) It partially inhibited the
`binding of anti-Leu-3a to CD4+ T cells. (iii) It reacted with
`HIV antigens in commercial HTL V-III and LAV ELISAs.
`(iv) It reacted by viable membrane immunofluorescence
`assay with HIV-infected human T cells but not uninfected
`cells. (v) It bound to a molecule of Mr 110,000-120,000 in
`immunoblot analysis of HIV-infected-cell lysate. (vi) It
`bound a recombinant gp160 peptide by a double-antibody
`radioimmunoassay (RIA). (vii) The binding of anti-Leu-3a to
`its anti-Id mAb was inhibited by mAbs against CD4 but not
`by irrelevant mAbs. (viii) It partially neutralized HIV infec(cid:173)
`tion of human T cells in vitro. These results strongly suggest
`that mAb HFl. 7 reacts with an idiotypic (Id) determinant on
`anti-Leu-3a and mimics part(s) of the CD4 molecule that
`represents the viral receptor for HIV and binds to HIV
`envelope glycoprotein. This binding may prevent the virus
`from attaching to target cells, resulting in viral neutralization.
`mAb HFl.7 may be an important reagent in the understand(cid:173)
`ing of the molecular mechanism of HIV pathogenicity and in
`the development of diagnostic and therapeutic strategies.
`
`MATERIALS AND METHODS
`mAbs. The CD4-specific mAbs anti-Leu-3a (Becton
`Dickinson), OKT4A (Ortho Diagnostics), and anti-T4 (Coul(cid:173)
`ter Immunology) were purchased from their manufacturer as
`purified immunoglobulins or were the gift of G. Thorton
`(Johnson and Johnson Biotechnology Center, La Jolla, CA).
`mAbs that recognize other lymphocyte phenotypic markers
`(Leu-1, Leu-2a, Leu-Sb, Leu-8, Leu-Ml) were purchased as
`purified immunoglobulins from Becton Dickinson.
`Generation of Monoclonal Anti-Id Antibodies. Three- to
`five-week-old BALB/c mice were immunized intravenously
`with purified anti-Leu-3a mAb (30 µ,g per mouse) in 0.9%
`NaCL Six injections were given at weekly intervals. Three
`days after the last injection, the mice were killed and their
`spleen cells were fused with the mouse myeloma cell line
`NS-1 as described previously (9). Supernatant fluids from
`wells with hybrid growth were screened for reactivity against
`HIV or anti-Leu-3a by an ELISA described below.
`ELISAs. The HTLV-III ELISA (Electro-Nucleonics,
`Silver Spring, MD) and the LAV EIA (Genetic Systems,
`Seattle, WA) were done according to the manufacturers'
`specifications. Horseradish peroxidase-conjugated goat anti(cid:173)
`mouse lgG antibodies (Vector Laboratories, Burlingame,
`CA) were substituted for goat anti-human lgG enzyme
`
`Abbreviations: AIDS, acquired immunodeficiency syndrome; FITC,
`fluorescein isothiocyanate; HIV, human immunodeficiency virus;
`Id, idiotype (idiotypic); mAb, monoclonal antibody; SV40Tantigen,
`simian virus 40 large tumor antigen; TCID~, 50% tissue culture
`infective dose.
`*To whom reprint requests should be addressed at: Department of
`Virology and Immunology, Southwest Foundation for Biomedical
`Research, P.O. Box 28147, San Antonio, TX 78284.
`
`3891
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`3892 Medical Sciences: Chanh et al.
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`Proc. Natl. Acad. Sci. USA 84 ( 1987)
`
`Table 1. Reactivity of mAb HFl. 7 with HIV antigens in ELISA
`
`HTLV-III ELISA
`
`LAV EIA
`
`mAb
`Negative control
`anti-Id
`0.04 ± 0.01
`0.06 ± 0.01
`0.05 ± 0.01
`Pooled AIDS serum•
`1.20 ± 0.11
`1.01 ± 0.10
`1.45 ± 0.15
`HFl.7 anti-Id
`0.45 ± 0.03
`1.20 ± 0.10
`0.75 ± 0.08
`Each value represents the mean± SEM of triplicate determinations. See Materials and Methods for
`descriptions of the assays.
`*Diluted 1:300.
`
`Psoralen- and UV-
`inactivated HIV
`
`conjugate. The ELISA using psoralen- and UV-inactivated
`HIV was done as described (10).
`To determine the binding of HFl. 7 to anti-Leu-3a, ascites
`fluid containing HFl. 7 or a control anti-Id mAb (GB-2, which
`recognizes an idiotype associated with a mAb specific for
`hepatitis B surface antigen) was fractionated with 50%(cid:173)
`saturated ammonium sulfate. The resulting immunoglobulin(cid:173)
`containing precipitate was resuspended in borate-buffered
`saline (0.05 M, pH 8.2), and the concentration of antibody
`was determined, using an extinction coefficient of 14fora1 %
`solution at 280 nm. Various concentrations of the anti-Id
`mAbs were adsorbed to triplicate wells of microtiter plates.
`After nonspecific sites were blocked by incubation with 10%
`normal goat serum in borate-buffered saline, either biotinyl(cid:173)
`ated anti-Leu-3a or a biotinylated control mAb specific for
`simian virus 40 large tumor antigen (SV 40 T antigen) (11) was
`added. (The antibodies had been biotinylated at a concen(cid:173)
`tration of7 mg/ml, and a 1:1000 dilution in 10% normal goat
`serum was used in the assay.) After a 1-hr incubation at 37°C,
`unbound antibodies were removed by washing, and specific
`binding was detected by using avidin-horseradish peroxidase
`and followed by 2,2'-azinobis(3-ethylbenzthiazolinesulfonic
`acid) (ABTS) with H20 2 • This assay was performed accord(cid:173)
`ing to methods previously described (12).
`Inhibition of Binding of Anti-Id mAb HFl. 7 to mAb Anti(cid:173)
`Leu-3a. Microtiter plates were coated with purified HFl. 7
`(500 ng per well). After blocking of nonspecific sites, 5 µ,g of
`various inhibitors were added to the anti-Id-coated wells for
`1 hr. After incubation and washing to remove unbound
`antibodies, biotinylated anti-Leu-3a at a 1:1000 dilution was
`added and the ELISA was done as described above.
`Immunofluorescence Staining. The immunofluorescence
`staining procedure was performed essentially as described
`(13). In brief, 106 cells were incubated with anti-Id HFl.7 or
`a negative antibody control of the same isotype for 30 min at
`4°C, followed by fluorescein isothiocyanate (FITC)-conju(cid:173)
`gated goat anti-mouse lgG (Cappel Laboratories, Cochran(cid:173)
`ville, PA) for an additional 30 min at 4°C. After incubation,
`
`1.0
`
`A
`
`0.8
`
`B
`
`the cells were washed, fixed in 0.37% formaldehyde, and
`analyzed by flow cytometry using a Becton Dickinson F ACS
`analyzer interfaced to a BD Consort 30 (Becton Dickinson).
`To assess the inhibition of binding of anti-Leu-3a to CD4 +
`cells by HFl.7, the human T-cell line CEM A3.0l was used
`(14). FITC-anti-Leu-3a (Becton Dickinson) was incubated
`with phosphate-buffered saline (PBS: O.OZ M, pH 7.4) or with
`PBS containing purified HFl.7 or control anti-Id mAb (10 µ,g)
`for 1hrat4°C and then was added to 5 x 105 A3.0l cells. The
`cells were incubated for 30 min at 4°C, washed twice, and
`analyzed on the FACS.
`Immunoblot Analysis. The Bio-Rad Immunoblot System
`(Bio-Rad Laboratories) was used. In brief, nitrocellulose
`strips on which electrophoretically fractionated HIV antigens
`had been blotted were incubated in 20 mM Tris·HCl/150 mM
`NaCl, pH 7.4/1% bovine serum albumin/0.2% Tween 20 to
`block nonspecific sites. The strips then were treated with
`pooled human AIDS sera (1:100) or 3-fold concentrated
`hybridoma supematants containing anti-Id antibodies over(cid:173)
`night at 4°C. The strips were washed with Tris·HCI buffer to
`remove unbound antibodies. Human and mouse antibody
`reactivities were detected with alkaline phosphatase-conju(cid:173)
`gated goat anti-human immunoglobulin and anti-mouse im(cid:173)
`munoglobulin (Sigma), respectively. The substrate used was
`provided by Bio-Rad Laboratories.
`Binding to Recombinant IDV Envelope Antigens. A recom(cid:173)
`binant gpl60 peptide produced in the baculovirus expression(cid:173)
`vector system and p\Jrified by lectin chromatography (Micro
`Gene Sys, West Haven, CT) was radiolabeled with 1251 by the
`chloramine-T reaction (15). Unreacted 1251 was removed by
`passage through a PD-10 column (Pharmacia). Approximate(cid:173)
`ly 92% of the radiolabel precipitated with protein in 10%
`trichloroacetic acid. A double-antibody RIA, similar to
`methods described in ref. 16, was performed using a hyper(cid:173)
`immune rabbit anti-mouse lgG to precipitate all the mouse
`lgG that bound the 1251-labeled gpl60.
`Neutralization of HIV Infection in Vitro. The neutralization
`assay was done as described (17). In brief, 1000or100 TCID50
`
`0.6
`
`0 ;;
`0
`0 0.4
`
`0.2
`
`0
`
`5.0
`
`2.0
`
`1.0 0.5
`
`5.0
`0.1
`0.2
`Anti-idiotype, µ.g
`
`2.0
`
`1.0 0.5
`
`0.2
`
`0.1
`
`FIG. 1. Binding ofbiotinylated
`anti-Leu-3a to anti-Id mAb HFl.7.
`Microtiter wells were coated with
`various amounts of HFL 7 mAb
`(•)or GB-2 control anti-Id mAb of
`the same isotype (o) and treated
`with biotinylated anti-Leu~3a (A)
`or biotinylated antibodies to SV 40
`T antigen (B).
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`2 of 5
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`Proc. Natl. Acad. Sci. USA 84 (1987)
`
`3893
`
`(see below for definition) of HIV in 100 µ1 was incubated with
`100 µl ofHFl.7 or GB-2 control anti-Id or culture medium for
`1 hr at 37°C. The concentrations of mAbs were adjusted to
`yield a final concentration of 0.5 mg/ml. After incubation,
`the treated HIV were added to 106 A3.0l cells and incubated
`at 37°C for 2 hr in the presence of Polybrene (Calbiochem) at
`10 µg/ml. The cells were then washed and resuspended (106
`per ml) in RPMI 1640 medium supplemented with 10% fetal
`bovine serum. At various times, aliquots of culture fluids
`were removed and reverse transcriptase (RNA-directed
`DNA polymerase, EC 2.7.7.49) activity was determined as
`described (17). Cell-free HIV was harvested from infected
`A3.0l cell culture and titrated on uninfected A3.0l cells, and
`the titer was expressed as 50% tissue culture infective dose
`(TCIDso).
`
`RESULTS
`Because our primary goal was to obtain mAbs reactive with
`HIV antigens, we chose to screen the hybrids by ELISA with
`HIV antigen-coated plates (Table 1). Among 389 hybrids
`tested, two were found that reacted in all three assays used.
`Thirty-five hybrids reacted with the immunizing antigen,
`mAb anti-Leu-3a (data not shown). One of the two hybrid(cid:173)
`omas producing mAbs reactive with HIV antigens, designat(cid:173)
`ed HFl. 7, was cloned twice by limiting dilution. The isotype
`of mAb HFl. 7 was determined to be lgM.
`To assess the specificity of HFl.7 binding, microtiter
`plates were coated with various concentrations of HFl. 7 or
`a control mAb, GB-2, and allowed to react with biotinylated
`anti-Leu-3a (Fig. lA) or biotinylated control mAb of the same
`isotype as anti-Leu-3a but recognizing SV40 T antigen (Fig.
`lB). Anti-Id mAb HFl. 7 specifically bound to the biotinyl(cid:173)
`ated anti-Leu-3a, whereas no binding was observed between
`the biotinylated anti-Leu-3a and the control anti-Id mAb.
`Neither HFl. 7 nor the control anti-Id mAb bound to biotinyl(cid:173)
`ated control mAb specific for SV40 T antigen. Anti-Id HFl.7
`did not react with a panel of irrelevant murine mAbs that
`included anti-Leu-1, -Leu-2a, -Leu-5b, -Leu-8, and -Leu-Ml
`or with normal mouse lgG.
`At a concentration of 5 µg, the irrelevant mAbs failed to
`significantly inhibit the binding of anti-Leu-3a to its anti-Id
`mAb (range of inhibition 0-5%; Table 2). On the other hand,
`anti-Leu-3a and two other mAbs that recognize the CD4
`molecule (0KT4A and anti-T4) were efficient inhibitors of
`the Id-anti-Id reaction. These data indicate that HFl.7
`recognizes an Id determinant on anti-Leu-3a and that it may
`"mimic" CD4 in its binding to anti-CD4 mAbs. It is note(cid:173)
`worthy that anti-Leu-3a, OKT4A, and anti-T4 all block in
`vitro infection by HIV (18). Thus, the ability to inhibit the
`Id-anti-Id reaction appears to correlate with the ability of the
`mAb to block HIV infection in vitro.
`
`Inhibition of binding of HFl.7 to anti-Leu-3a by
`Table 2.
`various antibodies
`
`Isotype
`IgGl,K
`IgGl,K
`IgGl,K
`IgG2a,K
`IgGl,K
`IgG2a,K
`IgG2a,K
`IgM,K
`
`Inhibitor
`Percent inhibition*
`Anti-Leu-3a
`94
`OKT4A
`91
`Anti-T4
`84
`Anti-Leu-1
`0
`Anti-Leu-2a
`0
`Anti-Leu-Sb
`4
`s
`Anti-Leu-8
`Anti-Leu-Ml
`3
`s
`Normal mouse IgGt
`Each inhibitor was tested at a concentration of S µ.g per well.
`*Mean of triplicate determinations.
`tPurified from pooled normal BALB/c mouse serum.
`
`200
`
`~
`Q)
`..0
`E
`~ z
`Q) 100
`u
`
`Q)
`>
`:;:
`0
`Q; a::
`
`0
`0
`10
`
`I
`
`10
`
`2
`10
`
`3
`10
`
`Relative Fluorescence Intensity
`Inhibition of binding of FITC-anti-Leu-3a to A3.0l cells
`FIG. 2.
`by anti-Id mAb HFl.7. The A3.01 cells were stained with FITC(cid:173)
`anti-Leu-3a in the presence of PBS (trace A) or PBS containing 10 µ.g
`of HFl.7 (trace B) or 10 µ.g of GB-2 (trace C).
`
`The binding of mAb HFl.7 to anti-Leu-3a was further
`confirmed in another inhibition experiment using flow cy(cid:173)
`tometry. Approximately 95% of cells of the human T-cell line
`A3.0l express surface CD4 as detected by immunofluores(cid:173)
`cence staining with anti-Leu-3a (14). Incubation of anti-Leu-
`3a with the HFl. 7 anti-Id mAb resulted in a significant
`decrease in the fluorescence intensity of the anti-Leu-3a
`staining (Fig. 2). Anti-Leu-3a staining of the A3.0l cells was
`not significantly affected by prior incubation with the control
`anti-Id mAb. These data suggest that the anti-Id mAb can
`bind to anti-Leu-3a and partially inhibit anti-Leu-3a binding
`to surface CD4 present on human T cells. Therefore, the
`anti-Id mAb must recognize at least a portion of the antibody(cid:173)
`combining site on anti-Leu-3a, based on its ability to inhibit
`binding to CD4 on human T cells. These characteristics
`further suggest that HFl. 7 recognizes an Id determinant
`associated with the antibody-combining site on anti-Leu-3a.
`To assess the expression of the antigen recognized by
`HFl.7 on the surface of HIV-infected cells by the anti-Id, an
`indirect immunofluorescence assay was performed on
`uninfected and continuously infected H9 cells (Fig. 3).
`Anti-Id staining of infected H9 cells resulted in a clear
`increase in fluorescence intensity, whereas uninfected H9
`cells were not stained. Approximately 25% of HIV-infected
`
`~
`Q)
`.0
`E
`~ z
`Q) 100
`u
`
`Q)
`>
`~
`0 a;
`a::
`
`Relative Fluorescence Intensity
`Immunofluorescence profiles of uninfected (trace A) and
`FIG. 3.
`HIV-infected (trace B) H9 cells stained with mAb HFl.7. Trace C
`shows GB-2 (negative control) staining of HIV-infected H9 cells.
`
`3 of 5
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`3894 Medical Sciences: Chanh et al.
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`2 3
`
`120--88-
`
`65-
`
`55 -
`
`42-
`
`24-
`
`18-
`
`Fto. 4.
`lmmunoblot analysis of HIV-infected cells. Blots were
`probed with pooled human AIDS serum diluted 1:100 (lane 1), mAb
`GB-2 (negative control; lane 2), or mAb HFl.7 (lane 3). Molecular
`weight markers (M, x io- 3) are shown at left.
`
`Proc. Nari. Acad. Sci. USA 84 ( 1987)
`
`fluid, 41% of the gp160 was bound with the anti-Id mAb. The
`control anti-Id preparation, GB-2, bound only 6% of the
`ml-labeled gp160 at a similar dilution of ascites. Excess
`unlabeled gp160 (10 µ,g) inhibited the binding of the HFl.7
`mAb to 12.Sl-labeled gpl60 by >95% (data not shown). These
`data indicate that the anti-Id mAb HFl.7 can bind the
`envelope glycoprotein of HIV.
`The ability of HFl. 7 mAb to inactivate HIV was assessed
`in an in vitro neutralization assay described previously (17).
`HIV replication was determined by measuring the reverse
`transcriptase activity in the culture supernatant fluids (Table
`3). Reverse transcriptase activity was inhibited in cultures
`treated with HFl.7 anti-Id in a viral·dose-dependent fashion.
`The most pronounced inhibition of viral replication was
`observed on day 7 of culture, when 58% and 90% inhibition
`of reverse transcriptase activity was observed with 1000 and
`100 TCID50 of HIV, respectively. By day 9 of culture, the
`reduction of reverse transcriptase activity in HFl. 7 treated
`cultures declined to 44% and 80% with 1000 and 100 TCID50
`of HIV, respectively. In contrast, GB-2-treated cultures
`produced approximately the same reverse transcriptase ac(cid:173)
`tivity as that detected in medium-treated cultures. The
`increased reverse transcriptase activity in cultures treated
`with HFl. 7 mAb on day 9 of culture presumably resulted
`from replication of HIV that escaped inactivation.
`
`DISCUSSION
`The causative agent of AIDS, HIV, primarily infects target
`cells that express the CD4 molecule. Antibodies, such as
`anti-Leu-3a and OKT4A, directed against the CD4 molecule
`effectively block the in vitro infectivity of HIV, presumably
`by competing with viral receptors. By utilizing anti-Leu-3a as
`the immunogen and selecting the resulting antibodies based
`on their ability to bind HIV antigens, we have generated an
`anti-Id mAb termed HFl.7, which appeared to "mimic" the
`CD4 determinant(s) involved in binding to mv. HFl.7 was
`specific for anti-Leu-3a; it did not bind to any of a panel of
`mouse mAbs with different specificities or to normal mouse
`IgG. HFl.7 recognized an Id determinant closely associated
`with the binding site of anti-Leu-3a, since it effectively
`blocked the binding of anti-Leu-3a to cells of the human
`T-cell line A3.0l, 9.5% of which express the CD4 molecules.
`In viable-cell-membrane immunofluorescence assays, mAb
`HFl.7 bound to .... 25% of HIV-infected H9 cells but not to
`uninfected cells; this observation suggests that the antigenic
`determinant detected by HFl. 7 is a component of the HIV
`envelope and that it is exposed at the surfac.e of infected
`lymphocytes.
`Although no direct evidence is available to indicate that
`HFl.7 anti-Id bears an internal image, the observations that
`it (iJ hound to anti-Leu-3a but not to irrelev(lltt mouse mAbs,
`(ii) inhibited the binding of anti-Leu-3a to CD4, (iii) recog(cid:173)
`nized an HIV envelope antigen with an approximate M, of
`110,000-120,000, and (iv) recognized a common Id shared by
`anti-CD4 mAbs that block HIV replication in vitro make it
`reasonable to speculate that the HFl.7 anti-Id bears an
`internal image that mimics the HIV viral receptor, the CD4
`molecule. Radioimmunoprecipitation studies (7) have dem-
`
`H9 cells were stained by the HFl. 7 anti-Id. To determine the
`kinetics of the surface expression of the antigen recognized
`by the anti-Id on in vitro HIV-infected cells, we infected the
`human T-cell line A3.0l with HIV isolate NY-5 (19) and
`pcrfonned a viable-cell-membrane indirect immunofluores(cid:173)
`cence assay with anti-Id mAb on day 1 to day 7 of infection.
`The antigen recognized by HFI. 7 was not detected until day
`4 of infection, at which point 10-15% of the A3 .01 cells were
`stained (data not shown). Thus, the anti-Id appears to
`recognize a determinant(s) present on HIV infected T cells.
`To characterize the antigen reactive wilh HFl. 7 anti-Id, we
`exposed nitrocellulose paper strips (Bio-Rad lmmunoblot
`Assay), on which HIV antigens had been electroblotted, to
`HFl.7 mAb or to the negative control anti-Id. A pooled
`human AIDs serum was used as a positive control, at a
`dilution of 1:100. The human antisera recognized the char(cid:173)
`acteristic HIV gag proteins p18 and p24 and the gag precursor
`p55 in addition to the envelope glycoproteins gp120 and gp41
`(Fig. 4). HFI.7 anti-Id reacted with a band corresponding to
`gpl20, with an approximate M, between 110,000 and 120,000.
`No reactivity was found with the negative mAb control. The
`anti-Id recognized the HIV envelope glycoprotein gp120,
`which appears to represent the region where HIV binds the
`CD4 molecule.
`To confirm the immunoblot analysis, a recombinant gp160
`peptide produced in baculovirus was radiolabeled, and the
`percentage of this antigen that could be bound by the HFl. 7
`mAb was determined. At a 1:40 dilution of delipidated ascites
`Tatlle 3. Neutralization of HIV infection in vitro by mAb HFl.7
`Revene transcriptase activity,• cpm
`
`Virus
`concentration,
`Day 7 of infection
`Day 9 of infection
`TCID50
`Medium
`GB-2
`HFl.7
`Medium
`GB-2
`HFl.7
`1000
`31,S62
`30,110 (S)
`12,760 (SS)
`160,156
`161,0SS (0)
`91,026 (43)
`100
`4,094
`3,569 (13)
`369 (91)
`S4,516
`53,476 (2)
`10,836 (80)
`*Each value represents the mean of duplicate cultures (see ref. 17 for reverse transcriptase assay). Numbers in parentheses indicate percent
`reduction in activity detennined as [(cpm in medium alone - cpm in the presence of antibody)lcpm in medium alone] x 100.
`
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`Celltrion, Inc., Exhibit 1105
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`Medical Sciences: Chanh et al.
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`Proc. Natl. Acad. Sci. USA 84 (1987)
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`3895
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`onstrated binding of CD4 to a HIV envelope glycoprotein
`molecule of M, 110,000.
`Although the in vitro neutralization of HIV infectivity by
`HFl. 7 was not complete, at least with the doses of HIV and
`anti-Id employed, these studies suggest that an internal(cid:173)
`image anti-Id that mimics the viral receptor for HIV on
`susceptible T cells can partially inhibit viral replication. It is
`noteworthy that an anti-Id mAb recognizes only a single
`antigenic determinant on the viral envelope and may not be
`efficient at completely neutralizing viral infectivity. Similar(cid:173)
`ly, the anti-Id mAb bound only 41% of a recombinant gp160
`protein. These facts also suggest that the affinity of this
`anti-Id mAb for HIV antigens may be low. A pool of several
`anti-Id mAbs that recognize several sites on the viral enve(cid:173)
`lope or a polyclonal anti-Id response may be more efficient in
`inhibiting viral replication and specific binding to the enve(cid:173)
`lope glycoprotein. Recently, it was shown (20) that rabbit
`polyclonal anti-Id antibodies against anti-CD4 mAbs failed to
`bind HIV or inhibit the binding of the anti-CD4 mAbs to
`CD4+ T cells. These polyclonal anti-Id antibodies appeared
`to recognize noncombining-site private Id expressed only on
`the anti-CD4 mAb utilized as an immunogen. This kind of
`anti-Id antibody has been referred to as an Ab-2,,, rather than
`the internal-image type of anti-Id antibody, referred to as
`Ab-2,s (21), that we describe here.
`Numerous studies have demonstrated that anti-Id antibod(cid:173)
`ies can mimic various substances and bind biological recep(cid:173)
`tors (for a review, see ref. 22). More important and relevant
`to this report is that anti-Id antibodies have been used to
`isolate and identify the mammalian reovirus receptor (23, 24)
`and to identify receptors that may bind the envelope glyco(cid:173)
`protein gp70 from murine leukemogenic retroviruses (25).
`The anti-Id antibody that recognized the reovirus receptor
`was capable of neutralizing viral infection of neurons (26).
`Based on the previous studies, it appears reasonable to utilize
`anti-Id antibody that can mimic a receptor, such as CD4, and
`bind a virus (HIV) at the site on the virus where it interacts
`with its receptor. This binding to HIV by the anti-Id antibody
`might be expected to neutralize infectivity by blocking the
`viral sites of attachment to the receptor.
`In addition, studies reviewed in refs. 27 and 28 have
`indicated the possible role of anti-Id as vaccines against
`infectious agents. Recently, the vaccine potential for anti-Id
`was demonstrated for hepatitis B virus in chimpanzees, the
`relevant animal model for human infection (29). Because the
`anti-Id described in the present report partially neutralized
`HIV infection in vitro, one might speculate that the induction
`of a polyclonal anti-Id response elicited by anti-Leu-3a
`immunization could represent a possible means for vaccina(cid:173)
`tion against HIV. The studies described herein demonstrate
`that an anti-Id can be produced that mimics the viral receptor
`for HIV and binds the virus. This binding of the anti-Id to
`HIV can inhibit viral replication in vitro. Such reagents may
`be useful in understanding the molecular mechanisms of HIV
`pathogenicity. Anti-Id may also be used to develop new
`strategies for diagnosis of HIV infection.
`
`We thank B. Alderete, M. Dookhan, and E. Reed for expert
`technical assistance. The NY-5 strain of HIV was a gift from Dr. T.
`Folks, National Institute of Allergy and Infectious Diseases,
`Bethesda, MD. The purified baculovirus-produced gp160 was the gift
`ofDrs. Gale Smith, Mark Cochrane, and Brad Erickson (Micro Gene
`Sys, West Haven, CT). This work was supported by New Investi(cid:173)
`gator Award AI22307 and Grants AI23619, AI23472, and HL32505
`from the National Institutes of Health and by contract DAMD
`17-86-C-6290 from the U.S. Army Research and Development
`Command.
`
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`5 of 5
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`Celltrion, Inc., Exhibit 1105
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