`
`").:,
`
`· ····~
`
`JUNE 1987
`VOLUME84
`NUMBER 11
`
`. .
`
`,r. '. ~ES
`
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`
`TU
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`
`Proceedings
`
`OF THE
`
`National Acaden1y
`of Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`BIOEPIS EX. 1105
`Page 1
`
`
`
`Proceedings
`OF THE
`National Academy
`of Sciences
`OF THE UNITED STATES OF AMERICA
`
`Officers
`of the
`Academy
`
`Editorial Board
`of the
`Proceedings
`
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`
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`GoRDON A. BA YM
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`
`MAXINE F. SINGER, Chairman
`MARY-DELL CHILTON
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`PETER D. LAX
`DANIEL NATHANS
`
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`
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`BIOEPIS EX. 1105
`Page 2
`
`
`
`Proc. Nat/. A cad. Sci. USA
`Vol. 84, pp. 3891-3895, June 1987
`Medical Sciences
`
`MonoclonaJ anti-idiotypic antibody mimics the CD4 receptor and
`binds human immunodeficiency virus
`
`(acquired immunodeficiency syndrome/ receptor mimicry IT-lymphocyte surface molecule)
`TRAN c. CI-IANH, GORDON R. DREESMAN , AND RONALD c. KENNEDY *
`Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, TX 78284
`
`Communicated by A/fred Nisonoff, February 20, 1987
`
`A monoclonal anti-idiotypic (anti-Id) anti(cid:173)
`ABSTRACT
`body, HF1.7, was generated against anti-Leu-3a, a mouse
`monoclonal antibody (mAb) specific for the CD4 molecule on
`baman helper/inducer T lymphocytes. The ant_i-Id nature of
`HF1.7 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
`tbe binding of anti-Leu-3a to CD4+ T cells. (iii) It detected a
`common idiotype present on various anti-CD4 mAbs. Because
`tbe 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-ld mAb HF1.7 to mimic CD4 and bind HIV. This
`uti-Id mAb reacted with HIV antigens in commercial HIV
`ELISAs and recognized HIV -infected human T cells but not
`aninfected cells when analyzed by flow cytofluoronietry. At(cid:173)
`testing further to the HIV specificity, the anti-Id mAb reacted
`with a recombinant gpl60 peptide and a molecule of M,
`110,000-120,000 in immunoblot 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
`tbat this anti-Id mAb mimics the CD4 antigenic determinants
`involved in binding to HIV.
`
`Acquired immunodeficiency syndrpme (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 (HTL V-III), lymphadenopa(cid:173)
`thy-associated virus (LA V) , and AIDS-associated retrovirus
`(ARV)] , the etiological agent of AibS, is lymphotropic for
`cells expressing the CD4 molecule. HIV has been shown to
`infe~t not only the helper/ inducer subset ofT lymphocytes
`but also cells of the monocyte/ macrophage lineage (1-4). In
`vitro infection by HIV can be effectiveiy 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 Mr 110,000. Tll.ese results imply
`that the CD4 antigenic determina.nts tecognized by anti-Leu-
`3a and OKT4A either represent the site of attachment ofHIV
`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 glycoproteirl . This interaction in turn may inhibit
`the binding of HIV to CD4 on target cells and therefore could
`lead to viral inactivation.
`A monoclonal anti-Id antibody, termed HF1.7, was gen(cid:173)
`erated against mAb anti-Leu-3a. HFl. 7 exhibited the follow(cid:173)
`ing pn:ipehies. (i) It reacted in solid-phase enzyme-linked
`
`The publication costs of this 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.
`
`3891
`
`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 LA V 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.
`tnAb HF1.7 may be an important reagent in the understand·
`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 arid Johnson Biotechnology Center, La Jolla, CA).
`mAbs that recognize other lymphocyte phenotypic markers
`(Leu-1, Leu-2a, Leu-5b, Leu-8, Leu-Ml) were purchased as
`purified immunoglobulins from llecton 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 p.g per mouse) in 0.9%
`NaCI. 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 ah ELISA described below.
`ELlS As. The HTL V-III ELISA (Electro-Nucleonics ,
`Silver Spring, MD) and the LA V EIA (Genetic Systems ,
`Seattle, WA) were done according to the manufacturers'
`specifications. Horseradish peroxidase-conjugated goat anti(cid:173)
`mouse IgG antibodies (Vector Laboratories , Burlingame,
`CA) were substituted for goat anti-human IgG enzyme
`
`Abbreviations : AIDS , acquired immunodeficiency syndrome ; FITC ,
`fluorescein isothiocyanate ; HIV , human immunodeficiency virus;
`ld , idiotype (idiotypic); mAb , monoclonal antibody ; SV40 T antigen ,
`simian virus 40 large tumor antigen ; TCID50, 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.
`
`BIOEPIS EX. 1105
`Page 3
`
`
`
`3892
`
`Medical Sciences: Chanh et al .
`
`Proc. Nat/. A cad. Sci. USA 84 ( 1987)
`
`Table 1. Reactivity of mAb HF1.7 with HIV antigens in ELISA
`
`HTLV-III ELISA
`
`LAY EIA
`
`mAb
`Negative control
`anti-Id
`0.04 ± 0.01
`0.06 ± 0.01
`0.05 ± 0.01
`1.01 ± 0.10
`1.45 ± 0.15
`1.20 ± 0.11
`Pooled AIDS serum*
`0.45 ± 0.03
`Q.75 ± 0.08
`HF1.7 anti-Id
`1.20 ± 0.10
`Each value represents the mean ± SEM of triplicate determinations. See Materials and M ethods 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 HF1.7 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 14 for a 1%
`solution at 280 nm. Various concentrations of tqe anti-ld
`mAbs were adsorbed to triplicate wells of microtiter plates.
`After nonspecific sites were blocked by incubation wit\1. 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 HF1.7 to mAb Anti(cid:173)
`Leu-3lj.. Microtiter plates were coated with purified HFl. 7
`(500 ng per weB). After blocking of nonspecific sites, 5 J.Lg of
`various inhibitors were aqded 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 HF1.7 or
`a negative antibody control of the same jsotype for 30 min at
`4°C, followed by fluore scein isothiocyanate (FITC)-conju(cid:173)
`gated goat anti-mouse IgG (Cappel Laboratories, Cochran(cid:173)
`ville, PA) for an additional 30 min at 4°C. After incubation,
`
`A
`
`1.0
`
`0 .8
`
`8
`
`the cells were washed , fixed in 0.37% formaldehyde, and
`analyzed by flow cytometry using a Becton Dickinson FACS
`analyzer interfaced to a J3D Consort 30 (Becton Dickinson).
`To assess the inhibition of binding of anti-Lel!-3a to CD4+
`cells by HF1.7 , the human T-cellline CEM A3.01 was used
`(14). FITC-anti-Leu-3a (Becton Dickinson) was incubated
`with phosphate-buffered saline (PBS: 0.02 M, pH 7.4) or with
`PBS containing purified HF1.7 or control anti-ld mAb (10 ~g)
`for 1 hr at 4°C and then was added to 5 x 105 A3 .01 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 wl)ich electrophoretically fractionated HIV antigens
`had been blotted were incubate9 in 20 mM Tris·HCI/150 mM
`NaCI , pH 7.4/ 1% bovine serum albumin/ 0.2% Tween 20 to
`block nonspecific sites . The striN then were treated with
`pooled human AIDS sera (1 :100) or 3-folq concentrated
`hybridoma StJpernatants containing anti-Id antibodies over(cid:173)
`night at 4°C. The strips were washed with Tris·HCl buffer to
`remove unbound antibodies. Human and mouse antibody
`reactivities were detected with alkaline phosphatase-conju(cid:173)
`gated goat anti-human immunoglob4lin 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 gp160 peptide produced in the baculovirus expression(cid:173)
`vector system and purified by lectin chromatography (Micro
`Gene Sys, West Haven , CT) was radiolabeled with 1251 by the
`chloramine-T reaction (15). U nreacted 125I was removed by
`passage through a PD-10 column (Pharmacja) . 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 IgG to precipitate all the mouse
`lgG that bound the 125I-labeled gp160.
`·
`Neutralization of HIV Infection in Vitro. The neutralization
`assay was done as described (17). In brief, 1000 or 100 TCIDso
`
`0 .6
`
`0
`
`0
`0 0.4
`
`0 .2
`
`0
`
`5.0
`
`2 .0
`
`1.0 0 .5
`
`0.2
`5.0
`0.1
`Anti-idiotype, JL9
`
`2 .0
`
`1.0
`
`0 .5
`
`0 .2 0 .1
`
`FlG. 1. Binding ofbiotinylated
`anti-Leu-3a to anti-Id mAb HF1.7.
`Microliter wells were coated with
`various amounts of HF1.7 mAb
`(e ) or GB-2 contrql anti-Id mAb of
`the same isotype (0) and treated
`with biotinylated anti-Leu-3a (A)
`or biotinylated antibodies to SV40
`T antigen (B).
`
`BIOEPIS EX. 1105
`Page 4
`
`
`
`Medical Sciences: Chanh et al.
`
`Proc. Nat/. A cad. Sci. USA 84 ( 1987)
`
`3893
`
`below for definition) ofHIV in 100 JLl was incubated with
`ofHF1.7 or GB-2 control anti-Id or culture medium for
`37°C. The concentrations of mAbs were adjusted to
`a final concentration of 0.5 mg/ ml. After incubation,
`HIV were added to 106 A3.01 cells and incubated
`2 hr in the presence ofPolybrene (Calbiochem) at
`The cells were then washed and resuspended (106
`in RPMI 1640 medium supplemented with 10% fetal
`serum. At various times, aliquots of culture fluids
`removed and reverse transcriptase (RNA-directed
`polymerase, EC 2.7.7.49) activity was determined as
`(17). Cell-free HIV was harvested from infected
`culture and titrated on uninfected A3.01 cells, and
`was expressed as 50% tissue culture infective dose
`
`RESULTS
`our primary goal was to obtain mAbs reactive with
`antigens, we chose to screen the hybrids by ELISA with
`antigen-coated plates (Table 1). Among 389 hybrids
`two were found that reacted in all three assays used.
`hybrids reacted with the immunizing antigen,
`anti-Leu-3a (data not shown). One of the two hybrid(cid:173)
`producing mAbs reactive with HIV antigens, designat(cid:173)
`.7, was cloned twice by limiting dilution. The isotype
`HF1.7 was determined to be IgM.
`assess the specificity of HFl. 7 binding, microtiter
`were coated with various concentrations of HF1. 7 or
`mAb, GB-2, and allowed to react with biotinylated
`(Fig. 1A) or biotinylated control mAb ofthe same
`as anti-Leu-3a but recognizing SV40 T antigen (Fig.
`Anti-Id mAb HF1.7 specifically bound to the biotinyl(cid:173)
`anti-Leu-3a, whereas no binding was observed between
`lbit:>ti11ylate:d anti-Leu-3a and the control anti-Id mAb.
`. 7 nor the control anti-Id mAb bound to biotinyl(cid:173)
`mAb specific for SV40 T antigen. Anti-Id HF1.7
`with a panel of irrelevant murine mAbs that
`anti-Leu-1 , -Leu-2a, -Leu-5b, -Leu-8, and -Leu-M1
`normal mouse IgG.
`concentration of 5 JLg, the irrelevant mAbs failed to
`inhibit the bindmg of anti-Leu-3a to its anti-Id
`of inhibition 0-5%; Table 2). On the other hand,
`3a and two other mAbs that recognize the CD4
`(OKT4A and anti-T4) were efficient inhibitors of
`Id reaction. These data indicate that HFl. 7
`an Id determinant on anti-Leu-3a and that it may
`" CD4 in its binding to anti-CD4 mAbs. It is note-
`that anti-Leu-3a, OKT4A, and anti-T4 all block in
`mte:cticm by HIV (18). Thus, the ability to inhibit the
`reaction appears to correlate with the ability of the
`to block HIV infection in vitro.
`
`IgGl ,K
`IgGl ,K
`lgG1,K
`lgG2a,K
`IgGl,K
`lgG2a,K
`lgG2a,K
`lgM,K
`
`94
`91
`84
`0
`0
`4
`5
`3
`5
`was tested at a concentration of 5 J.Lg per well.
`triplicate determinations.
`from pooled normal BALB/ c mouse serum.
`
`200
`
`.._
`a.>
`.D
`E
`::J z
`a.> 100
`u
`a.>
`>
`:;:
`.2
`a.> a::
`
`0
`0
`10
`
`I
`
`10
`
`2
`10
`
`3
`10
`
`Relative Fluorescence Intensity
`Inhibition of binding of FITC-anti-Leu-3a to A3 .01 cells
`FIG. 2.
`by anti-ld mAb HF1.7. The A3.01 cells were stained with FITC(cid:173)
`anti-Leu-3a in the presence of PBS (trace A) or PBS containing 10 J.Lg
`of HF1.7 (trace B) or 10 J.Lg of GB-2 (trace C).
`
`The binding of mAb HF1.7 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
`A3.01 express surface CD4 as detected by immunofluores(cid:173)
`cence staining with anti-Leu-3a (14). Incubation of anti-Leu-
`3a with the HF1.7 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 A3.01 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)
`combming 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
`HF1.7 on the surface ofHIV-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
`
`.._
`a.>
`.0
`E
`::J z
`a.> 100
`u
`a.>
`>
`
`~ a.> a::
`
`I 10
`Relative Fluorescence Intensity
`Immunofluorescence profiles ofuninfected (trace A) and
`FIG. 3.
`HIV-infected (trace B) H9 cells stained with mAb HF1.7 . Trace C
`shows GB-2 (negative control) staining of HIV-infected H9 cells.
`
`BIOEPIS EX. 1105
`Page 5
`
`
`
`3894 Medical Sciences: Chanh et al.
`
`Proc. Nat/. Acad. Sci. USA 84 (1 987)
`
`2 3
`
`120-
`88-
`
`65-
`
`55-
`
`42-
`
`24-
`
`18-
`
`Immunoblot analysis of HIV-infected cells . Blots were
`FIG. 4.
`probed with pooled human AIDS serum diluted 1:100 (lane 1), mAb
`GB-2 (negative control; lane 2) , or mAb HF1.7 (lane 3). Molecular
`weight markers (Mr x 10- 3) are shown at left.
`
`H9 cells were stained by the HF1.7 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 A3.01 with HIV isolate NY-5 (19) and
`performed 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 HF1.7 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 with HF1.7 anti-Id, we
`exposed nitrocellulose paper strips (Bio-Rad Immunoblot
`Assay), on which HIV antigens had been electroblotted, to
`HF1.7 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). HF1.7 anti-Id reacted with a band corresponding to
`gp120, with an approximate Mr 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 HF1. 7
`mAb was determined. At a 1:40 dilution of delipidated ascites
`
`fluid , 41% of the gp160 was bound with the anti-Id mAb. The
`control anti-Id preparation, GB-2, bound only 6% of the
`125I-labeled gp160 at a similar dilution of ascites. Excess
`unlabeled gp160 (10 J.Lg) inhibited the binding of the HFl 7
`mAb to 125I-labeled gp160 by > 95% (data not shown). The~e
`data indicate that the anti-Id mAb HF1.7 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 TCID.10
`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 HF1.7 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-3aas
`the immunogen and selecting the resulting antibodies based
`on their ability to bind HIV antigens , we have generated an
`anti-Id mAb termed HF1.7 , which appeared to "mimic" the
`CD4 determinant(s) involved in binding to HIV. HF1.7 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-cellline A3 .01, 95% of which express the CD4 molecules.
`In viable-cell-membrane immunofluorescence assays, mAb
`HF1.7 bound to ""25% of HIV-infected H9 cells but not to
`uninfected cells; this observation suggests that the antigeni~
`determinant detected by HFl. 7 is a component of the HI
`envelope and that it is exposed at the surface of infected
`lymphocytes.
`Although no direct evidence is available to indicate that
`HF1.7 anti-Id bears an internal image , the observations that
`it (i) bound to anti-Leu-3a but not to irrelevant mouse mAbs,
`(ii) inhibited the binding of anti-Leu-3a to CD4, (iii) recogf
`
`nized an HIV envelope antigen with an approximate Mr:
`110,000-120,000, and (iv) recognized a common Id shared ~
`anti-CD4 mAbs that block HIV replication in vitro make 1
`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 deJJI·
`
`_T_ab_l_e_3_. __ N_e_u_tr_ali_·z_a_ti_o_n_o_f_H_I_V_i_n~_e_c_ti_on __ in_v_i_rr_o_b~y_mA ___ b_H_F_1_.7 _______________________________________________ ____
`___________________________ R_e_v_e_rs_e_t_ran __ sc_n~· p~t_as_e_a_c_ti_v,_·ty~,_*_c~p_m _______________________ ____
`
`Day 7 of infection
`
`Virus
`concentration,
`TCIDso
`
`Medium
`
`GB-2
`
`HF1.7
`
`-----
`Day 9 of infection
`------------~--------------~
`~
`Medium
`GB-2
`91,026 (43))
`80
`161 ,058 (0)
`160,156
`12,760 (58)
`30,110 (5)
`31,562
`1000
`10~
`53,476 (2)
`54 ,516
`369 (91)
`3,569 (13)
`4,094
`100
`*Each value represents the mean of duplicate cultures (see ref. 17 for reverse transcriptase assay) . Numbers in parentheses indicate percent
`reduction in activity determined as [(cpm in medium alone - cpm in the presence of antibody)/cpm in medium alone] x 100.
`
`BIOEPIS EX. 1105
`Page 6
`
`
`
`binding of CD4 to a HIV envelope glycoprotein
`of Mr 110,000.
`AllnUII~u the in vitro neutralization of HIV infectivity by
`was not complete, at least with the doses of HIV and
`employed, these studies suggest that an internal(cid:173)
`anti-Id that mimics the viral receptor for HIV on
`1CC:vuu1c T cells can partially inhibit viral replication. It is
`••um,.,thv that an anti-Id mAb recognizes only a single
`determinant on the viral envelope and may not be
`at completely neutralizing viral infectivity. Similar(cid:173)
`Id mAb bound only 41% of a recombinant gpl60
`These facts also suggest that the affinity of this
`mAb for HIV antigens may be low. A pool of several
`mAbs that recognize several sites on the viral enve(cid:173)
`a polyclonal anti-ld response may be more efficient in
`viral replication and specific binding to the enve-
`glycoJlro1tein. Recently, it was shown (20) that rabbit
`anti-ld antibodies against anti-CD4 mAbs failed to
`or inhibit the binding of the anti-CD4 mAbs to
`. These polyclonal anti-Id antibodies appeared
`~clgnize noncombining-site private ld expressed only on
`au1.a-..... v .. mAb utilized as an immunogen. This kind of
`·ldant:ibcldy has been referred to as an Ab-2a, rather than
`IDt,erntat-Im:age type of anti-Id antibody, referred to as
`we describe here.
`have demonstrated that anti-Id antibod(cid:173)
`various substances and bind biological recep(cid:173)
`a review, see ref. 22). More important and relevant
`report is that anti-Id antibodies have been used to
`and identify the mammalian reovirus receptor (23, 24)
`identify receptors that may bind the envelope glyco-
`gp70 from murine leukemogenic retroviruses (25).
`ld antibody that recognized the reovirus receptor
`of neutralizing viral infection of neurons (26).
`the previous studies, it appears reasonable to utilize
`antibody that can mimic a receptor, such as CD4, and
`virus (HIV) at the site on the virus where it interacts
`receptor. This binding to HIV by the anti-Id antibody
`be expected to neutralize infectivity by blocking the
`sites of attachment to the receptor.
`addition, studies reviewed in refs. 27 and 28 have
`the possible role of anti-ld as vaccines against
`agents. Recently, the vaccine potential for anti-ld
`lllelrnonsttralted for hepatitis B virus in chimpanzees, the
`animal model for human infection (29) . Because the
`described in the present report partially neutralized
`infection in vitro, one might speculate that the induction
`polyclonal anti-ld response elicited by anti-Leu-3a
`IIDllation could represent a possible means for vaccina(cid:173)
`. The studies described herein demonstrate
`!I'll iWil-10 can be produced that mimics the viral receptor
`and binds the virus. This binding of the anti-Id to
`inhibit viral replication in vitro. Such reagents may
`in understanding the molecular mechanisms of HIV
`DRe:nic~itv . Anti-ld may also be used to develop new
`for diagnosis of HIV infection.
`
`thank B. Alderete, M. Dookhan, and E. Reed for expert
`assistance. The NY -5 strain of HIV was a gift from Dr. T.
`Institute of Allergy and Infectious Diseases,
`. The purified baculovirus-produced gp160 was the gift
`Smith, Mark Cochrane, and Brad Erickson (Micro Gene
`Haven, CT). This work was supported by New Investi(cid:173)
`AthWard ~I22307 and Grants AI23619, AI23472 , and HL32505
`e NatiOnal Institutes of Health and by contract DAMD
`from the U.S. Army Research and Development
`
`Proc. Nat/. Acad. Sci. USA 84 (1987)
`
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