Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2546-2550, April 1992 Immunology
Anti-idiotype monoclonal antibody elicits broadly neutralizing anti-gpl20 antibodies in monkeys (anti-idiotype antibody/neutralizing antibody/human immunodeficiency virus vaccine)
CHANG-YUIL KANG*t, PETER NARAt, SOULAIMA CHAMAT*, VINCE CARALLI*, AGNES CHEN*, MAI-LAN NGUYEN*, HIRONORI YOSHIYAMA§, W. J. W. MORROW*, DAVID D. Ho§, AND HEINZ K6HLER* *IDEC Pharmaceuticals Corporation, La Jolla, CA 92037; *The National Cancer Institute, Frederick, MD 21701; and §The Aaron Diamond AIDS Research Center, New York, NY 10016
Communicated by Alfred Nisonoff, December 26, 1991 (received for review October 17, 1991)
CD4 attachment site of gpl20. Targeting such B cells with specific anti-idiotype (or anti-clonotype) antibodies may stimulate the synthesis of specific antibodies. In this regard, we utilized broadly neutralizing human polyclonal anti-gpl20 antibodies as template idiotype antibody (Abl) to generate murine anti-idiotype monoclonal antibodies (Ab2 mAbs), since it was believed that this approach will increase the chance of generating anti-anti-idiotype antibodies (Ab3) in humans that are close to Abls in terms of clonality and specificity. One anti-idiotype mAb, 3C9, that interacted with CD4 site-specific, broadly neutralizing antibodies was selected and tested in cynomolgus monkeys. The data indicate that this anti-idiotype mAb induced broadly neutralizing anti-gp120 antibodies in nonhuman primates.
Murine monoclonal antibodies (mAbs) were ABSTRACT raised against human, polyclonal, anti-gpl20 antibodies (Abl) and were selected for binding to broadly neutralizing antigpl20 antibodies in sera positive for human immunodeficiency virus (HIV). One anti-idiotype mAb (Ab2), 3C9, was found to be specific for human anti-gpl20 antibodies directed against an epitope around the conserved CD4 attachment site of gpl20. The 3C9 reactive human anti-gpl20 antibodies (3C9' Ab) neutralized MN, huB, RF, and four primary isolates of HIV type 1 (HIV-1). Cynomolgus monkeys were immunized with 3C9 in adjuvant to test whether this anti-idiotype mAb could induce neutralizing anti-gpl20 antibodies. The results show that purified anti-anti-idiotype antibodies (Ab3) from 3C9 immune sera bind to an epitope around the CD4 attachment site of gpl2OSF and gpl2OmII. Furthermore, purified gpl20specific Ab3 neutralize MN, fIB, and RF isolates. These results demonstrate that primates immunized with an antiidiotype mAb produce broadly neutralizing anti-HIV-1 antibodies. Since this anti-idiotype mAb was selected by identifying a clonotypic marker, its biological activity can be explained as the result of clonotypic B-cell stimulation.
MATERIALS AND METHODS Immunizations. Four cynomolgus monkeys received intramuscular injections with 2.5 mg of 3C9 in 1 ml of SAF (Syntex adjuvant formulation) containing 0.6 mg of N-acetylmuramyl-L-alanyl-D-isoglutamine. The injections were administered biweekly. Animals were bled before the injection series and 7 days after each injection. Recombinant Proteins. gpl20IIIB is a recombinant glycoprotein of a HIV111B isolate that is secreted from a Drosophila cell line. The gpl2Om1B was received from SmithKline Beecham. gp120sF2 is a recombinant envelope glycoprotein of an HIVs5 isolate that is secreted from Chinese hamster ovary (CHO) cells. gp120sF2 was received from Chiron. Recombinant soluble CD4 (sCD4) was prepared by transfection of a truncated CD4 gene into CHO cells. Secreted sCD4 in culture supernatants of CHO cells (representing 95% of the amino-terminal extracellular portion of the molecule) was purified on an anti-CD4 antibody-conjugated Sepharose column. Affinity Purification of Antibodies. All affinity columns were prepared by conjugating 8 mg of protein with 5 ml of CNBr-activated Sepharose 4B (Pharmacia). To purify the 3C9-reactive antibody (3C9' Ab) from pooled sera of healthy HIV-infected individuals (6), heat (560C for 1 hr)- and detergent (1% Nonidet P40)-treated sera were applied to a 3C9conjugated Sepharose affinity column. After being washed extensively, bound antibodies (3C9' Abs) were eluted with citrate buffer (pH 2.8) and dialyzed against phosphatebuffered saline (PBS; pH 7.2). To purify various Ab3s from monkey sera, all 3C9 immune sera were initially diluted 1:1 with PBS. The diluted serum was then passed over an affinity column of normal mouse immunoglobulin-conjugated Seph-
The immunodominant neutralizing site against human immunodeficiency virus type 1 (HIV-1) resides in the third hypervariable region (V3) of gpl20. The hypervariability of the amino acid composition outside and within this region suggests that HIV may be able to escape the effects of V3specific neutralizing antibodies by mutation (1). The second major neutralizing epitope of gpl20 is the CD4 attachment site. A recent report has demonstrated the conformational dependence of this region (2). Furthermore, our study has shown that several epitopes reside around the CD4 attachment site (3). Antibodies specific for these epitopes generally exhibit broad neutralizing activities against multiple strains of HIV-1 (2-5). Thus, the CD4 attachment site of gpl20 is an attractive target for immunotherapy as the problems of antigenic variation may be minimized. However, the natural antibody response to this region is weaker, at least in early infection (1, 2), and the neutralizing activities of the CD4 site-specific antibodies are less potent than that of V3specific antibodies (6). In the present study, we explored the use of anti-idiotype antibodies, to induce CD4 site-directed, broadly neutralizing antibodies in nonhuman primates. The reason for using this approach was to overcome the limiting immunogenic potential of the CD4 epitopes. We believed that the inherent antigenically imposed restriction associated with virus-based vaccines could be overcome by using antibodies directed against idiotopes on B-cell receptors with specificity for the
Abbreviations: Abl, idiotype antibody; Ab2, anti-idiotype antibody; Ab3, anti-anti-idiotype antibody; V3, the third variable region of gpl20; sCD4, soluble CD4; mAb, monoclonal antibody; BSA, bovine serum albumin. tTo whom reprint requests should be addressed at: IDEC Pharmaceuticals Corporation, 11099 North Torrey Pines Road, La Jolla, CA 92037.
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.
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Proc. Natl. Acad. Sci. USA 89 (1992)
arose. The flow-through fraction was collected and set aside. The bound antibody (anti-isotype Ab) was washed, eluted with citrate buffer, and dialyzed against PBS. The flowthrough fraction from the above purification was then passed over the 3C9-Sepharose affinity column. The bound antibody (Ab3) was washed, eluted with citrate buffer, and dialyzed against PBS. Subsequently, the Ab3 was passed over a
gpl20sF-Sepharose affinity column, and the column
was
washed extensively. The bound antibody (gpl20-specific Ab3) was eluted with citrate buffer and dialyzed against PBS. RIA. gpl20SF2 (Chiron) and gp120I11B (SmithKline Beecham) were labeled with 1251 by using a Bolton-Hunter reagent kit (NEN). RIA plates (Dynatech) were coated for 18 hr with various amounts of antibody in PBS. The plates were washed and then blocked for 1 hr with PBS containing 1% bovine serum albumin (BSA). 125I-labeled gp120jI1B or gp120sF2 in PBS containing 10%o (vol/vol) fetal calf serum and 0.05% Tween 20 was added in the presence or absence of different concentrations of inhibitors. After 3 hr, the plates were washed with PBS, and the bound radioactivity was determined in a y counter. ELISA. To test the binding activity of 3C9' Abs to a V3 region-derived peptide (amino acids 301-338 of gp120s2), microtiter plates were coated overnight with 0.25 pkg of protein in PBS per well. After the adherent layer was blocked and washed, different concentrations of antibody in PBS were added to the plates. Alkaline phosphatase-coupled goat anti-human immunoglobulin and phosphatase substrate (Sigma) were used to detect antibodies binding to the plates. Neutralization Assays. Two quantitative neutralization assays with HIV-1 laboratory strains were performed as described (7, 8). Briefly, monolayers of CEM-SS target cells were cultured with different HIV strains in the absence or presence of different neutralizing antibodies. After 3-5 days in culture, syncytia-forming units were counted, and in the same assay, supernatants were assayed in ELISA for p24 concentration 7-12 days later. Neutralization assays with HIV primary isolates were performed as described (9). RESULTS
Property of Anti-Idiotype mAb 3C9 as Candidate for Clonotypic Stimulation. Recently, we generated a series of antiidiotypic antibodies against purified human anti-gpl20 antibodies from a pool of four healthy HIV-1-infected donors (3). Briefly, anti-gpl20 antibodies were purified on gp1205s2Sepharose and were used to immunize mice for hybridoma fusion. Anti-idiotype mAbs were selected for binding to human anti-gpl20 antibodies. One anti-idiotype mAb, 3C9, with specificity for CD4 site-directed anti-gpl20 antibodies, was
further characterized as
a
candidate for induction of
anti-gpl20 antibodies. Pooled human HIV-1+ sera were purified on a 3C9 immunoabsorbent, and eluted antibodies (3C9+ Ab) were tested for binding to gpl20 and V3 loop peptides. The results (Table 1) show that 3C9+ Ab bound to two genetically distinct gpl20s derived from HIVs5 and HIVIIIB and did not bind to the V3 loop peptide. Furthermore, 3C9+ Ab binding to gpl20 was inhibited by sCD4, indicating that 3C9+ Ab is specific for an epitope around the CD4 attachment site of gpl20. Next, the biological function of 3C9+ Ab was examined in virus neutralization assays. Human 3C9+ Ab exhibited different spectra of broadly neutralizing activities against genetically distinct HIV laboratory strains (Fig. 1 Left) and four primary isolates established from HIV-infected individuals (Fig. 1 Right). Collectively, these results indicate that anti-idiotype mAb 3C9 recognizes a clonotypic determinant shared by human polyclonal antigpl20 antibodies that are specific for a conserved epitope around the CD4 attachment site and exhibit broadly neutralizing activities. By this definition, 3C9 defines a marker on B cells producing neutralizing anti-gpl20 antibodies.
2547
Table 1. Immunological properties of human 3C9-reactive antibodies (3C9' Ab) Binding activity of 3C9' Ab in assays cpm in RIA with 1251_gp120t
gpl20SF2 gp1205~ ~ gp120IHlB l2OI~lB
A405 in
ELISA with Without With Without With V3 loop Ab* sCD4 sCD4 sCD4 sCD4 peptidef 3C9+ Ab 5058 1539 8394 0.07 1963 Normal hIg 145 151 282 278 1.77 V3-specific hAb (polyclonal) *3C9+ Ab was purified from HIV+ human sera on a 3C9-conjugated Sepharose column, normal human immunoglobulin (hIg) was from The Binding Site (San Diego), and V3-specific human polyclonal antibodies (hAb) were purified from HIV+ human sera on a V3SF2conjugated Affi-Gel column as described (6). tPlates were coated with 0.3 1g of antibody per well. After addition of PBS containing 10%6 fetal calf serum to block the adsorbed layer, 5 x 104 cpm of 125I-labeled gp12OsF2 or 125I-labeled gp120jII1 was added to each well in the presence or absence of sCD4 at 50 ,ug/ml. After 3 hr, the plates were washed and the radioactivity was measured. *Plates were coated with 0.25 ,ug of V3SF2 peptide in PBS per well. After addition of PBS containing 1% BSA to block the adsorbed layer, 1 ,ug of antibodies per ml of PBS was added to the plates. Alkaline phosphatase-coupled goat anti-human Ig and phosphatase substrate were used to detect antibodies binding to the plates. Absorbance was measured at 405 nm.
3C9 mAb Induces Anti-gpl20 Ab3 Antibodies. To test whether 3C9 elicits anti-gpl20 antibodies, four cynomolgus monkeys were immunized with 3C9 mAb in Syntex adjuvant formulation. Since the immune serum was expected to contain 3C9-specific antibodies (Ab3) and mouse immunoglobulin-specific antibodies (anti-isotype Ab), it was fractionated on successive affinity columns and tested for binding to gpl20. Ab3 from two monkeys bound to gpl20sF2 in an antibody concentration-dependent manner (Fig. 2 Left) and to gp120O1HB in an antigen concentration-dependent manner (Fig. 2 Right), whereas anti-isotype Abs did not bind to either type of gpl20. Similar results were obtained with Ab3s from two other monkeys (data not shown). Inhibition assays were performed to analyze the specificity of Ab3. The results in Table 2 show that gp120 binding of Ab3 from monkeys PRO703 and PRO783 were inhibited by 3C9 anti-idiotype mAb (Ab2), human 3C9+ Ab (Abl), and sCD4. Similar results were obtained with Ab3s from other monkeys (data not shown). These results confirm the anti-clonotypic nature of 3C9, as it is able to induce in primates gpl20-specific Ab3 with similar specificity to the human anti-gpl20 antibodies (3C9+ Ab) purified on 3C9 immunoabsorbent (see Table 1). Monkey Ab3 antibodies were further fractionated on a gp120sF2-Sepharose affinity column. The flow-through and bound fractions were collected as non-gpl20-specific Ab3 and gpl20-specific Ab3, respectively. These antibodies were then examined for gpl20 binding activity. The data in Table 3 show that all gpl20-specific Ab3 from four monkeys bound to gpl20, whereas non-gpl20-specific Ab3 did not. This finding indicates that only a fraction of Ab3 recognized gpl20 and also suggests that the gpl20-specific Ab3 from all four monkeys had a range of affinities for gpl20. For example, gpl20-specific Ab3 of PRO703 exhibited a 3- to 4-fold higher binding to gpl20 than that of gpl20-specific Ab3 from the other three monkeys. Neutralizing Activity of gpl20-Specific Ab3. Finally, we examined the HIV-1 virus-neutralizing activities of gpl20specific Ab3 antibodies. Table 4 summarizes the results. The gpl20-specific Ab3 from PRO703 neutralized MN, IIIB, and RF strains of HIV-1. The gpl20-specific Ab3 from PRO431
Proc. Natl. Acad. Sci. USA 89 (1992)
Immunology: Kang et al.
2548
c 0
c
0
4-
N
N
L.
= L.
4)
-
a)
z
z
Be
.1
1
10
0
0.04
0.13
Ab Conc.
Ab Conc. ,jLg/ml
0.4
1.2
3.6
,gg/ml
FIG. 1. Neutralizing activity of human 3C9' Ab against HIV laboratory strains (Left) and patient primary HIV isolates (Right). The neutralizing activity in Left is assessed by comparing the number of syncytial-forming units of virus in the well containing the test antibody with that in the control well. The neutralizing activity in Right was assessed by comparing the amount of p24 (a protein produced by HIV-1) in the well containing the test antibody with that in the control well.
observation of vigorous humoral and cell-mediated anti-HIV responses in healthy seropositive individuals. Accordingly, a realistic aim for AIDS therapy is the prolongation of the disease-free state of the infected individual by boosting and extending those immune reactions that control the spread of viral infection. Studies on offspring of infected mothers (10, 11) and passive antibody transfer experiments (12) suggest that neutralizing antibodies may play an important role in preventing virus infection or halting further virus spread. Conventional antigen preparations based on the envelope protein are bound by the epitope preference for the V3 loop as observed with the viral infection and vaccinations (1). The eventual failure of the antibody response to prevent continued virus infection is due to its focus on the immunodominant but hypervariable V3 loop epitopes, since emerging viral variants can escape the antibody (1). Although more conserved neutralizing epitopes such as the CD4 site exist (2-6), attempts to design vaccines based on peptides, recombinant
strongly neutralized the RF strain and weakly neutralized the MN and IIIB strains. The gpl20-specific Ab3 from PRO419 and PRO783 neutralized RF and MN, respectively. The lower or limited neutralizing activity of the gpl20-specific Ab3s from PRO419, PRO431, and PRO783 may be due to lower affinity against certain gpl20s. The gpl20 binding activity of gpl20-specific Ab3 from these monkeys was considerably lower than that ofgpl2O-specific Ab3 from PRO703 (Table 3). Collectively, the results from binding and virus neutralization studies on antibodies isolated from the sera of 3C9immunized nonhuman primates demonstrate that 3C9 can stimulate clones of primary B cells to produce broadly neutralizing HIV-1 antibodies.
DISCUSSION The prolonged course of HIV infection without clinical manifestation strongly suggests that immune mechanisms keep the virus under control. This notion receives support from the
500-
3000
r-
r
L
Q.
000 '
N U')
co
0
0
N
(N
0D
CM
500-
cl 0
0
co
0
0
01
10
Coated Ab Conc.,
4g/well
10
00
000
Added gpl2O0, CPM/well
FIG. 2. Binding of gpl2OSF2 (Left) and gp120OiIB (Right) to purified Ab3 from monkeys PRO703 and PRO783. For the gp120sF2 binding assay (Left), plates were coated with different concentrations of antibody. After blocking the adherent layer with 1% BSA in PBS, 1.5 x 105 cpm of 125I-labeled gp12OSF2 in PBS containing 10% fetal calf serum was added to the plates. After 3 hr, the plates were washed and the radioactivity was assayed. For the gpl20OjHB binding assay (Right), plates were coated with 1 pg of different antibodies per well. After the adherent layer was blocked with 1% BSA in PBS, different amounts of 125I-labeled gpl2OIIIB in PBS containing 10%o fetal calf serum were added to the plates. After 3 hr, the plates were washed and the radioactivity was measured. *, PRO703 Ab3; o, PRO703 anti-isotype Ab; *, PRO783 Ab3; o, PRO783 anti-isotype Ab.
Proc. Natl. Acad. Sci. USA 89 (1992)
Immunology: Kang et al. Table 2. Inhibition of Ab3 binding to gpl20sF2 by various inhibitors Monkey Ab3 binding Inhibitor Conc., jkg/ml % inhibition to gpl20sn 94 5 3C9 PRO703 Ab3 5 98 3C9+ Ab* 73 30t sCD4 2 100 BSA
PRO783 Ab3
95 5 3C9 5 97 3C9+ Ab* 79 30t sCD4 100 0 BSA Plates were coated with 2 pg of antibody per well. After addition of PBS containing 10%1 fetal calf serum to block the adsorbed layer, 1.5 x 105 of 125I-labeled gp12OSF2 was added to each well in the presence or absence of inhibitors. After 3 hr, the plates were washed, the radioactivity was assayed, and the percent inhibition was calculated. *3C9+ Ab was purified from HIV+ human sera on a 3C9-conjugated Sepharose column. tAt this concentration, sCD4 did not inhibit V3 region-specific antibody to gp120sF2, as previously described (3).
fragments, or intact gpl20 are limited because of poor immunogenicity (13) or the conformational nature of this site (2, 14). Since Nisonoff and Lamoyi proposed the potential use of anti-idiotype antibodies as vaccines (15), their utility has been demonstrated in several animal models (16, 17). The prevailing concepts behind this approach have been based on the structural mimicry of antigens by certain types of antiidiotype antibodies (Ab2,B) as theorized by Jerne et al. (18). However, there are several reports that indicate that certain Ab2s, not defined as Ab2p, can induce antigen-specific responses (19-21). To describe these biologically effective anti-idiotype antibodies, the term "network antigen" has been introduced to signify that such anti-idiotype antibodies are not internal-image Ab2s (22). Since 3C9 does not bind to CD4 (data not shown), it is unlikely that it represents the internal image of the CD4 attachment site on gpl20. In this regard, our approach in using 3C9 differs also from previous strategies based on presumed mimicry of the CD4 attachment site by anti-CD4 Table 3. Binding of gpl20-specific Ab3 antibodies to gpl20 Binding of 125I-labeled
gp120sF2 to Ab3, cpm Monkey PRO703 PRO783 PRO419
Ab3, jg per well 1 0.2
gpl20-specific Ab3 11,557
2,324
non-gpl20-specific Ab3 218 137
2 0.5
6,156
2,803
402 314
1 0.5 0.25
4,300 1,477 702
236 NT NT
212 1 5,738 NT 0.5 2,161 NT 0.25 1,100 For the binding assays, RIA plates were coated with different amounts of purified anti-gpl20 Ab3 in PBS for 18 hr. The plates were blocked with 1% BSA in PBS, and 1 x 10 cpm of125I-labeledgp12OsF2 was added to each well. After 3 hr, the plates were washed and the radioactivities were measured. NT, not tested.
PRO431
2549
Table 4. Neutralizing activity of gpl2O-specific Ab3 antibodies isolated from 3C9 immune primate sera Neutralizing activity as % inhibition of HIV isolates HIVnm Ab3, ug/ml Monkey HIVRF HIVmN 84 82 97 30 PRO703 94 20 97 10
PRO783
55 18.8
82 8
0 0
PRO419
30 15 7.5
10 8 13
20 0 9
ND ND 96
98 96
67 96 46 30 98 17 56 15 24 0 89 7.5 gpl20-specific Ab3 antibodies produced by monkey PRO703 were isolated from a pool of sera obtained after the second, third, fifth, and sixth immunizations. gpl2O-specific Ab3 antibodies from monkey PRO783 were isolated from a pool of sera obtained after the second, third, fifth, sixth, and seventh immunizations. Ab3 antibodies from monkeys PRO419 and PRO431 were isolated from a pool of sera obtained after the fourth and fifth immunizations. The neutralizing activity was assessed by comparing the amount of p24 in the well of the test antibody with that in the control well. All experiments were performed in triplicate. ND, not done.
PRO431
antibodies (23). Therefore, our scheme is based on the assumption that anti-idiotype antibodies that recognize a significant number of antigen-specific B-cell clones can be used to induce specific antibodies. An important feature of our approach is the concept of "idiotype repertoire matching" between the clonal B-cell repertoire targeted by antiidiotype antibodies and the antigen-specific antibody population that can interact with anti-idiotype antibodies. In other words, whether an anti-idiotype antibody can elicit antigenspecific immune responses depends on the existence of a common B-cell repertoire that recognizes antigen as well as anti-idiotype antibodies in target animals. This concept is different from the traditional view of anti-idiotype antibodies as structural antigen mimics, the so-called Ab2J3 population (18). Thus, an important criterion for selecting effective anti-idiotype antibodies is the degree of clonotypic representation in the host species. For example, when anti-idiotype antibodies are designed for human use, it is critical to utilize human antibodies as templates to generate anti-idiotype antibodies. This process should increase the chance of generating Ab3s in humans that exhibit antigen-binding potential similar to those of the template AbI antibodies. This hypothesis constitutes the basis for our use of polyclonal human neutralizing antibodies specific for the conserved CD4 site to generate anti-idiotype mAbs (3, 6). To define an Ab2 as a therapeutic vaccine candidate, the following criteria were used: (i) Ab2 should recognize human Abls that exhibit broadly neutralizing activities, and (ii) Ab2 should induce broadly neutralizing Ab3 in nonhuman primates. The similarities between 3C9-reactive human Abl and 3C9-induced monkey Ab3 can be summarized as follows: (i) both antibodies bind to gp12OsF2 and gpl20mB; (ii) they recognize an epitope around the CD4 attachment site; and (iii) they exhibit broadly neutralizing activities, although the neutralizing activities ofAb3 are limited and weaker than that of human Abl. Since certain Ab3s did not neutralize HIVmIIB strains despite their binding to gpl2OIIIB, we can speculate that the weaker or limited neutralizing activities of Ab3 may be due to a lower affinity against gpl2OIIIB. The relatively low affinity of gpl2O-specific Ab3 can be explained by the ab-
2550
Immunology: Kang et al.
sence of HIV antigen-primed B cells in immunologically naive monkeys. In contrast, it is anticipated that anti-idiotype antibodies will induce Ab3s of higher affinity in HIV-infected individuals who have preexisting, gp12O-primed, and affinitymatured B cells that can respond to stimulation by 3C9. Since 3C9 was not raised against monkey anti-HIV antibodies, we cannot expect a high degree of clonotypic representation in the naive B-cell repertoire of uninfected monkeys. Thus, it is not surprising to observe that the neutralizing response in the 3C9-immunized monkey is variable. Although the immunonaive monkey is not an ideal model for a HIVinfected human, we consider the data gained from the study of these animals to be very encouraging for developing therapeutic AIDS vaccines. We thank Drs. Nabil Hanna and Frank Norton for their helpful discussions, Nancy Dunlop for her technical assistance, Dr. Nancy Haigwood for providing gpl20, and Wendy Parker and Susette Fino for their assistance in the preparation of the manuscript. This study was supported in part by National Institutes of Health Grant 1R43AI31310 to C.-Y.K. and by the New York Life Insurance Company. 1. Nara, P. L., Garrity, R. D. & Goudsmit, J. (1991) FASEB J. 5, 2437-2455. 2. Ho, D. D., McKeating, J. A., Li, X. L., Moudgil, T., Daar, E. C., Sun, N.-C. & Robinson, J. E. (1991) J. Virol. 65, 489-493. 3. Chamat, S., Nara, P., Berquist, L., Whalley, A., Morrow, W. J. W., Kohler, H. & Kang, C.-Y. (1991) Modern Approaches to New Vaccines Including Prevention of AIDS: Vaccines 91 (Cold Spring Harbor Lab., Cold Spring Harbor, NY), p. 26 (abstr.). 4. Tilley, S. A., Honmen, W. J., Racho, M. E., Hilgartner, M. & Pinter, A. (1991) Res. Virol. 142, 247-259. 5. Posner, M. R., Hideshima, T., Cannon, T., Mukherjee, M., Mayer, K. H. & Byrn, R. (1991) J. Immunol. 146, 4325-4332. 6. Kang, C.-Y., Nara, P., Chamat, S., Caralli, V., Ryskamp, T., Haigwood, N., Newman, R. & Kohler, H. (1991) Proc. Natl. Acad. Sci. USA 88, 6171-6175. 7. Nara, P. L. & Fischinger, P. J. (1988) Nature (London) 332, 469-470. 8. Whalley, A. S., Nguyen, M.-L. & Morrow, W. J. W. (1991) Viral Immunol. 4, 201-213.
Proc. Natl. Acad. Sci. USA 89 (1992) 9. Daar, E. S., Li, X. L., Moudgil, T. & Ho, D. (1990) Proc. Natl. Acad. Sci. USA 87, 6574-6578. 10. Prince, M. A., Horowitz, B., Baker, L., Shulman, R. W., Ralph, H., Valinsk, J., Cundell, A., Brotman, B., Boehle, W., Rey, F., Piet, M., Reesink, A., Leslie, N., Tersmette, M., Miedema, F., Barbosa, L., Nemo, G., Nastala, C. L., Allan, J. S., Lee, D. R. & Eichberg, J. W. (1988) Proc. Natl. Acad. Sci. USA 85, 6944-6948. 11. Karpas, A., Hill, F., Youle, M., Cullen, V., Gray, J., Byron, N., Hayhoe, F. G. J., Tenant-Flowers, M., Howard, L., Gilgen, D., Oates, J. K., Hawkins, D. & Gazzard, B. (1988) Proc. Natl. Acad. Sci. USA 85, 9234-9237. 12. Emini, E. A., Schleif, W. A., Murthy, K., Eda, Y., Tokiyoshi, S., Putney, S. D., Matsushita, S., Nunberg, J. H. & Eichberg, J. W. (1991) Seventh International Conference on AIDS, Florence, TH.A.64 (Abstr.). 13. Haigwood, N. L., Nara, P. L., VanNest, G., Scandella, C. J., Eichberg, J. W. & Steimer, K. S. (1991) Modern Approaches to New Vaccines Including Prevention of AIDS: Vaccines 91 (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 51-58. 14. Morrow, W. J. W., Williams, W. M., Whalley, A. S., Ryskamp, T., Newman, R., Kang, C.-Y., Chamat, S., K6hler, H. & Kieber-Emmons, T. (1992) Immunology, in press. 15. Nisonoff, A. & Lamoyi, E. (1981) Clin. Immunol. Immunopathol. 44, 397-406. 16. Kennedy, R. C., Henkel, R. D., Pauletti, D., Allan, J. S., Lee, T. H., Essex, M. & Dreesman, G. R. (1986) Science 231, 1556-1559. 17. Kohler, H., Kaveri, S., Kieber-Emmons, T., Morrow, W. J. W., Muller, S. & Raychaudhuri, S. (1989) Methods Enzymol. 178, 3-35. 18. Jerne, N. K., Roland, J. & Cazenave, P.-A. (1982) EMBO J. 1, 243-245. 19. Schick, M. R., Dreesman, G. R. & Kennedy, R. C. (1987) J. Immunol. 138, 3419-3425. 20. Huang, J.-H., Ward, R. E. & Kohler, H. (1986) J. Immunol. 132, 770-776. 21. Kennedy, R. C., Dreesman, G. R., Butel, J. S. & Lanford, R. E. (1985) J. Exp. Med. 161, 1432-1449. 22. Kohler, H., Kieber-Emmons, T., Srinivasan, S., Kaveri, S., Morrow, W. J. W., Muller, S., Kang, C.-Y. & Raychaudhuri, S. (1989) Clin. Immunol. Immunopathol. 52, 104-116. 23. Healey, D. G., Dianda, L., Buch, D., Schroeder, K., Truneh, A., Sattentau, Q. J. & Beverly, P. C. (1991) Eur. J. Immunol. 21, 1491-1498.
Anti-idiotype monoclonal antibody elicits broadly neutralizing anti-gpl20 antibodies in monkeys (anti-idiotype antibody/neutralizing antibody/human immunodeficiency virus vaccine)
CHANG-YUIL KANG*t, PETER NARAt, SOULAIMA CHAMAT*, VINCE CARALLI*, AGNES CHEN*, MAI-LAN NGUYEN*, HIRONORI YOSHIYAMA§, W. J. W. MORROW*, DAVID D. Ho§, AND HEINZ K6HLER* *IDEC Pharmaceuticals Corporation, La Jolla, CA 92037; *The National Cancer Institute, Frederick, MD 21701; and §The Aaron Diamond AIDS Research Center, New York, NY 10016
Communicated by Alfred Nisonoff, December 26, 1991 (received for review October 17, 1991)
CD4 attachment site of gpl20. Targeting such B cells with specific anti-idiotype (or anti-clonotype) antibodies may stimulate the synthesis of specific antibodies. In this regard, we utilized broadly neutralizing human polyclonal anti-gpl20 antibodies as template idiotype antibody (Abl) to generate murine anti-idiotype monoclonal antibodies (Ab2 mAbs), since it was believed that this approach will increase the chance of generating anti-anti-idiotype antibodies (Ab3) in humans that are close to Abls in terms of clonality and specificity. One anti-idiotype mAb, 3C9, that interacted with CD4 site-specific, broadly neutralizing antibodies was selected and tested in cynomolgus monkeys. The data indicate that this anti-idiotype mAb induced broadly neutralizing anti-gp120 antibodies in nonhuman primates.
Murine monoclonal antibodies (mAbs) were ABSTRACT raised against human, polyclonal, anti-gpl20 antibodies (Abl) and were selected for binding to broadly neutralizing antigpl20 antibodies in sera positive for human immunodeficiency virus (HIV). One anti-idiotype mAb (Ab2), 3C9, was found to be specific for human anti-gpl20 antibodies directed against an epitope around the conserved CD4 attachment site of gpl20. The 3C9 reactive human anti-gpl20 antibodies (3C9' Ab) neutralized MN, huB, RF, and four primary isolates of HIV type 1 (HIV-1). Cynomolgus monkeys were immunized with 3C9 in adjuvant to test whether this anti-idiotype mAb could induce neutralizing anti-gpl20 antibodies. The results show that purified anti-anti-idiotype antibodies (Ab3) from 3C9 immune sera bind to an epitope around the CD4 attachment site of gpl2OSF and gpl2OmII. Furthermore, purified gpl20specific Ab3 neutralize MN, fIB, and RF isolates. These results demonstrate that primates immunized with an antiidiotype mAb produce broadly neutralizing anti-HIV-1 antibodies. Since this anti-idiotype mAb was selected by identifying a clonotypic marker, its biological activity can be explained as the result of clonotypic B-cell stimulation.
MATERIALS AND METHODS Immunizations. Four cynomolgus monkeys received intramuscular injections with 2.5 mg of 3C9 in 1 ml of SAF (Syntex adjuvant formulation) containing 0.6 mg of N-acetylmuramyl-L-alanyl-D-isoglutamine. The injections were administered biweekly. Animals were bled before the injection series and 7 days after each injection. Recombinant Proteins. gpl20IIIB is a recombinant glycoprotein of a HIV111B isolate that is secreted from a Drosophila cell line. The gpl2Om1B was received from SmithKline Beecham. gp120sF2 is a recombinant envelope glycoprotein of an HIVs5 isolate that is secreted from Chinese hamster ovary (CHO) cells. gp120sF2 was received from Chiron. Recombinant soluble CD4 (sCD4) was prepared by transfection of a truncated CD4 gene into CHO cells. Secreted sCD4 in culture supernatants of CHO cells (representing 95% of the amino-terminal extracellular portion of the molecule) was purified on an anti-CD4 antibody-conjugated Sepharose column. Affinity Purification of Antibodies. All affinity columns were prepared by conjugating 8 mg of protein with 5 ml of CNBr-activated Sepharose 4B (Pharmacia). To purify the 3C9-reactive antibody (3C9' Ab) from pooled sera of healthy HIV-infected individuals (6), heat (560C for 1 hr)- and detergent (1% Nonidet P40)-treated sera were applied to a 3C9conjugated Sepharose affinity column. After being washed extensively, bound antibodies (3C9' Abs) were eluted with citrate buffer (pH 2.8) and dialyzed against phosphatebuffered saline (PBS; pH 7.2). To purify various Ab3s from monkey sera, all 3C9 immune sera were initially diluted 1:1 with PBS. The diluted serum was then passed over an affinity column of normal mouse immunoglobulin-conjugated Seph-
The immunodominant neutralizing site against human immunodeficiency virus type 1 (HIV-1) resides in the third hypervariable region (V3) of gpl20. The hypervariability of the amino acid composition outside and within this region suggests that HIV may be able to escape the effects of V3specific neutralizing antibodies by mutation (1). The second major neutralizing epitope of gpl20 is the CD4 attachment site. A recent report has demonstrated the conformational dependence of this region (2). Furthermore, our study has shown that several epitopes reside around the CD4 attachment site (3). Antibodies specific for these epitopes generally exhibit broad neutralizing activities against multiple strains of HIV-1 (2-5). Thus, the CD4 attachment site of gpl20 is an attractive target for immunotherapy as the problems of antigenic variation may be minimized. However, the natural antibody response to this region is weaker, at least in early infection (1, 2), and the neutralizing activities of the CD4 site-specific antibodies are less potent than that of V3specific antibodies (6). In the present study, we explored the use of anti-idiotype antibodies, to induce CD4 site-directed, broadly neutralizing antibodies in nonhuman primates. The reason for using this approach was to overcome the limiting immunogenic potential of the CD4 epitopes. We believed that the inherent antigenically imposed restriction associated with virus-based vaccines could be overcome by using antibodies directed against idiotopes on B-cell receptors with specificity for the
Abbreviations: Abl, idiotype antibody; Ab2, anti-idiotype antibody; Ab3, anti-anti-idiotype antibody; V3, the third variable region of gpl20; sCD4, soluble CD4; mAb, monoclonal antibody; BSA, bovine serum albumin. tTo whom reprint requests should be addressed at: IDEC Pharmaceuticals Corporation, 11099 North Torrey Pines Road, La Jolla, CA 92037.
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.
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Proc. Natl. Acad. Sci. USA 89 (1992)
arose. The flow-through fraction was collected and set aside. The bound antibody (anti-isotype Ab) was washed, eluted with citrate buffer, and dialyzed against PBS. The flowthrough fraction from the above purification was then passed over the 3C9-Sepharose affinity column. The bound antibody (Ab3) was washed, eluted with citrate buffer, and dialyzed against PBS. Subsequently, the Ab3 was passed over a
gpl20sF-Sepharose affinity column, and the column
was
washed extensively. The bound antibody (gpl20-specific Ab3) was eluted with citrate buffer and dialyzed against PBS. RIA. gpl20SF2 (Chiron) and gp120I11B (SmithKline Beecham) were labeled with 1251 by using a Bolton-Hunter reagent kit (NEN). RIA plates (Dynatech) were coated for 18 hr with various amounts of antibody in PBS. The plates were washed and then blocked for 1 hr with PBS containing 1% bovine serum albumin (BSA). 125I-labeled gp120jI1B or gp120sF2 in PBS containing 10%o (vol/vol) fetal calf serum and 0.05% Tween 20 was added in the presence or absence of different concentrations of inhibitors. After 3 hr, the plates were washed with PBS, and the bound radioactivity was determined in a y counter. ELISA. To test the binding activity of 3C9' Abs to a V3 region-derived peptide (amino acids 301-338 of gp120s2), microtiter plates were coated overnight with 0.25 pkg of protein in PBS per well. After the adherent layer was blocked and washed, different concentrations of antibody in PBS were added to the plates. Alkaline phosphatase-coupled goat anti-human immunoglobulin and phosphatase substrate (Sigma) were used to detect antibodies binding to the plates. Neutralization Assays. Two quantitative neutralization assays with HIV-1 laboratory strains were performed as described (7, 8). Briefly, monolayers of CEM-SS target cells were cultured with different HIV strains in the absence or presence of different neutralizing antibodies. After 3-5 days in culture, syncytia-forming units were counted, and in the same assay, supernatants were assayed in ELISA for p24 concentration 7-12 days later. Neutralization assays with HIV primary isolates were performed as described (9). RESULTS
Property of Anti-Idiotype mAb 3C9 as Candidate for Clonotypic Stimulation. Recently, we generated a series of antiidiotypic antibodies against purified human anti-gpl20 antibodies from a pool of four healthy HIV-1-infected donors (3). Briefly, anti-gpl20 antibodies were purified on gp1205s2Sepharose and were used to immunize mice for hybridoma fusion. Anti-idiotype mAbs were selected for binding to human anti-gpl20 antibodies. One anti-idiotype mAb, 3C9, with specificity for CD4 site-directed anti-gpl20 antibodies, was
further characterized as
a
candidate for induction of
anti-gpl20 antibodies. Pooled human HIV-1+ sera were purified on a 3C9 immunoabsorbent, and eluted antibodies (3C9+ Ab) were tested for binding to gpl20 and V3 loop peptides. The results (Table 1) show that 3C9+ Ab bound to two genetically distinct gpl20s derived from HIVs5 and HIVIIIB and did not bind to the V3 loop peptide. Furthermore, 3C9+ Ab binding to gpl20 was inhibited by sCD4, indicating that 3C9+ Ab is specific for an epitope around the CD4 attachment site of gpl20. Next, the biological function of 3C9+ Ab was examined in virus neutralization assays. Human 3C9+ Ab exhibited different spectra of broadly neutralizing activities against genetically distinct HIV laboratory strains (Fig. 1 Left) and four primary isolates established from HIV-infected individuals (Fig. 1 Right). Collectively, these results indicate that anti-idiotype mAb 3C9 recognizes a clonotypic determinant shared by human polyclonal antigpl20 antibodies that are specific for a conserved epitope around the CD4 attachment site and exhibit broadly neutralizing activities. By this definition, 3C9 defines a marker on B cells producing neutralizing anti-gpl20 antibodies.
2547
Table 1. Immunological properties of human 3C9-reactive antibodies (3C9' Ab) Binding activity of 3C9' Ab in assays cpm in RIA with 1251_gp120t
gpl20SF2 gp1205~ ~ gp120IHlB l2OI~lB
A405 in
ELISA with Without With Without With V3 loop Ab* sCD4 sCD4 sCD4 sCD4 peptidef 3C9+ Ab 5058 1539 8394 0.07 1963 Normal hIg 145 151 282 278 1.77 V3-specific hAb (polyclonal) *3C9+ Ab was purified from HIV+ human sera on a 3C9-conjugated Sepharose column, normal human immunoglobulin (hIg) was from The Binding Site (San Diego), and V3-specific human polyclonal antibodies (hAb) were purified from HIV+ human sera on a V3SF2conjugated Affi-Gel column as described (6). tPlates were coated with 0.3 1g of antibody per well. After addition of PBS containing 10%6 fetal calf serum to block the adsorbed layer, 5 x 104 cpm of 125I-labeled gp12OsF2 or 125I-labeled gp120jII1 was added to each well in the presence or absence of sCD4 at 50 ,ug/ml. After 3 hr, the plates were washed and the radioactivity was measured. *Plates were coated with 0.25 ,ug of V3SF2 peptide in PBS per well. After addition of PBS containing 1% BSA to block the adsorbed layer, 1 ,ug of antibodies per ml of PBS was added to the plates. Alkaline phosphatase-coupled goat anti-human Ig and phosphatase substrate were used to detect antibodies binding to the plates. Absorbance was measured at 405 nm.
3C9 mAb Induces Anti-gpl20 Ab3 Antibodies. To test whether 3C9 elicits anti-gpl20 antibodies, four cynomolgus monkeys were immunized with 3C9 mAb in Syntex adjuvant formulation. Since the immune serum was expected to contain 3C9-specific antibodies (Ab3) and mouse immunoglobulin-specific antibodies (anti-isotype Ab), it was fractionated on successive affinity columns and tested for binding to gpl20. Ab3 from two monkeys bound to gpl20sF2 in an antibody concentration-dependent manner (Fig. 2 Left) and to gp120O1HB in an antigen concentration-dependent manner (Fig. 2 Right), whereas anti-isotype Abs did not bind to either type of gpl20. Similar results were obtained with Ab3s from two other monkeys (data not shown). Inhibition assays were performed to analyze the specificity of Ab3. The results in Table 2 show that gp120 binding of Ab3 from monkeys PRO703 and PRO783 were inhibited by 3C9 anti-idiotype mAb (Ab2), human 3C9+ Ab (Abl), and sCD4. Similar results were obtained with Ab3s from other monkeys (data not shown). These results confirm the anti-clonotypic nature of 3C9, as it is able to induce in primates gpl20-specific Ab3 with similar specificity to the human anti-gpl20 antibodies (3C9+ Ab) purified on 3C9 immunoabsorbent (see Table 1). Monkey Ab3 antibodies were further fractionated on a gp120sF2-Sepharose affinity column. The flow-through and bound fractions were collected as non-gpl20-specific Ab3 and gpl20-specific Ab3, respectively. These antibodies were then examined for gpl20 binding activity. The data in Table 3 show that all gpl20-specific Ab3 from four monkeys bound to gpl20, whereas non-gpl20-specific Ab3 did not. This finding indicates that only a fraction of Ab3 recognized gpl20 and also suggests that the gpl20-specific Ab3 from all four monkeys had a range of affinities for gpl20. For example, gpl20-specific Ab3 of PRO703 exhibited a 3- to 4-fold higher binding to gpl20 than that of gpl20-specific Ab3 from the other three monkeys. Neutralizing Activity of gpl20-Specific Ab3. Finally, we examined the HIV-1 virus-neutralizing activities of gpl20specific Ab3 antibodies. Table 4 summarizes the results. The gpl20-specific Ab3 from PRO703 neutralized MN, IIIB, and RF strains of HIV-1. The gpl20-specific Ab3 from PRO431
Proc. Natl. Acad. Sci. USA 89 (1992)
Immunology: Kang et al.
2548
c 0
c
0
4-
N
N
L.
= L.
4)
-
a)
z
z
Be
.1
1
10
0
0.04
0.13
Ab Conc.
Ab Conc. ,jLg/ml
0.4
1.2
3.6
,gg/ml
FIG. 1. Neutralizing activity of human 3C9' Ab against HIV laboratory strains (Left) and patient primary HIV isolates (Right). The neutralizing activity in Left is assessed by comparing the number of syncytial-forming units of virus in the well containing the test antibody with that in the control well. The neutralizing activity in Right was assessed by comparing the amount of p24 (a protein produced by HIV-1) in the well containing the test antibody with that in the control well.
observation of vigorous humoral and cell-mediated anti-HIV responses in healthy seropositive individuals. Accordingly, a realistic aim for AIDS therapy is the prolongation of the disease-free state of the infected individual by boosting and extending those immune reactions that control the spread of viral infection. Studies on offspring of infected mothers (10, 11) and passive antibody transfer experiments (12) suggest that neutralizing antibodies may play an important role in preventing virus infection or halting further virus spread. Conventional antigen preparations based on the envelope protein are bound by the epitope preference for the V3 loop as observed with the viral infection and vaccinations (1). The eventual failure of the antibody response to prevent continued virus infection is due to its focus on the immunodominant but hypervariable V3 loop epitopes, since emerging viral variants can escape the antibody (1). Although more conserved neutralizing epitopes such as the CD4 site exist (2-6), attempts to design vaccines based on peptides, recombinant
strongly neutralized the RF strain and weakly neutralized the MN and IIIB strains. The gpl20-specific Ab3 from PRO419 and PRO783 neutralized RF and MN, respectively. The lower or limited neutralizing activity of the gpl20-specific Ab3s from PRO419, PRO431, and PRO783 may be due to lower affinity against certain gpl20s. The gpl20 binding activity of gpl20-specific Ab3 from these monkeys was considerably lower than that ofgpl2O-specific Ab3 from PRO703 (Table 3). Collectively, the results from binding and virus neutralization studies on antibodies isolated from the sera of 3C9immunized nonhuman primates demonstrate that 3C9 can stimulate clones of primary B cells to produce broadly neutralizing HIV-1 antibodies.
DISCUSSION The prolonged course of HIV infection without clinical manifestation strongly suggests that immune mechanisms keep the virus under control. This notion receives support from the
500-
3000
r-
r
L
Q.
000 '
N U')
co
0
0
N
(N
0D
CM
500-
cl 0
0
co
0
0
01
10
Coated Ab Conc.,
4g/well
10
00
000
Added gpl2O0, CPM/well
FIG. 2. Binding of gpl2OSF2 (Left) and gp120OiIB (Right) to purified Ab3 from monkeys PRO703 and PRO783. For the gp120sF2 binding assay (Left), plates were coated with different concentrations of antibody. After blocking the adherent layer with 1% BSA in PBS, 1.5 x 105 cpm of 125I-labeled gp12OSF2 in PBS containing 10% fetal calf serum was added to the plates. After 3 hr, the plates were washed and the radioactivity was assayed. For the gpl20OjHB binding assay (Right), plates were coated with 1 pg of different antibodies per well. After the adherent layer was blocked with 1% BSA in PBS, different amounts of 125I-labeled gpl2OIIIB in PBS containing 10%o fetal calf serum were added to the plates. After 3 hr, the plates were washed and the radioactivity was measured. *, PRO703 Ab3; o, PRO703 anti-isotype Ab; *, PRO783 Ab3; o, PRO783 anti-isotype Ab.
Proc. Natl. Acad. Sci. USA 89 (1992)
Immunology: Kang et al. Table 2. Inhibition of Ab3 binding to gpl20sF2 by various inhibitors Monkey Ab3 binding Inhibitor Conc., jkg/ml % inhibition to gpl20sn 94 5 3C9 PRO703 Ab3 5 98 3C9+ Ab* 73 30t sCD4 2 100 BSA
PRO783 Ab3
95 5 3C9 5 97 3C9+ Ab* 79 30t sCD4 100 0 BSA Plates were coated with 2 pg of antibody per well. After addition of PBS containing 10%1 fetal calf serum to block the adsorbed layer, 1.5 x 105 of 125I-labeled gp12OSF2 was added to each well in the presence or absence of inhibitors. After 3 hr, the plates were washed, the radioactivity was assayed, and the percent inhibition was calculated. *3C9+ Ab was purified from HIV+ human sera on a 3C9-conjugated Sepharose column. tAt this concentration, sCD4 did not inhibit V3 region-specific antibody to gp120sF2, as previously described (3).
fragments, or intact gpl20 are limited because of poor immunogenicity (13) or the conformational nature of this site (2, 14). Since Nisonoff and Lamoyi proposed the potential use of anti-idiotype antibodies as vaccines (15), their utility has been demonstrated in several animal models (16, 17). The prevailing concepts behind this approach have been based on the structural mimicry of antigens by certain types of antiidiotype antibodies (Ab2,B) as theorized by Jerne et al. (18). However, there are several reports that indicate that certain Ab2s, not defined as Ab2p, can induce antigen-specific responses (19-21). To describe these biologically effective anti-idiotype antibodies, the term "network antigen" has been introduced to signify that such anti-idiotype antibodies are not internal-image Ab2s (22). Since 3C9 does not bind to CD4 (data not shown), it is unlikely that it represents the internal image of the CD4 attachment site on gpl20. In this regard, our approach in using 3C9 differs also from previous strategies based on presumed mimicry of the CD4 attachment site by anti-CD4 Table 3. Binding of gpl20-specific Ab3 antibodies to gpl20 Binding of 125I-labeled
gp120sF2 to Ab3, cpm Monkey PRO703 PRO783 PRO419
Ab3, jg per well 1 0.2
gpl20-specific Ab3 11,557
2,324
non-gpl20-specific Ab3 218 137
2 0.5
6,156
2,803
402 314
1 0.5 0.25
4,300 1,477 702
236 NT NT
212 1 5,738 NT 0.5 2,161 NT 0.25 1,100 For the binding assays, RIA plates were coated with different amounts of purified anti-gpl20 Ab3 in PBS for 18 hr. The plates were blocked with 1% BSA in PBS, and 1 x 10 cpm of125I-labeledgp12OsF2 was added to each well. After 3 hr, the plates were washed and the radioactivities were measured. NT, not tested.
PRO431
2549
Table 4. Neutralizing activity of gpl2O-specific Ab3 antibodies isolated from 3C9 immune primate sera Neutralizing activity as % inhibition of HIV isolates HIVnm Ab3, ug/ml Monkey HIVRF HIVmN 84 82 97 30 PRO703 94 20 97 10
PRO783
55 18.8
82 8
0 0
PRO419
30 15 7.5
10 8 13
20 0 9
ND ND 96
98 96
67 96 46 30 98 17 56 15 24 0 89 7.5 gpl20-specific Ab3 antibodies produced by monkey PRO703 were isolated from a pool of sera obtained after the second, third, fifth, and sixth immunizations. gpl2O-specific Ab3 antibodies from monkey PRO783 were isolated from a pool of sera obtained after the second, third, fifth, sixth, and seventh immunizations. Ab3 antibodies from monkeys PRO419 and PRO431 were isolated from a pool of sera obtained after the fourth and fifth immunizations. The neutralizing activity was assessed by comparing the amount of p24 in the well of the test antibody with that in the control well. All experiments were performed in triplicate. ND, not done.
PRO431
antibodies (23). Therefore, our scheme is based on the assumption that anti-idiotype antibodies that recognize a significant number of antigen-specific B-cell clones can be used to induce specific antibodies. An important feature of our approach is the concept of "idiotype repertoire matching" between the clonal B-cell repertoire targeted by antiidiotype antibodies and the antigen-specific antibody population that can interact with anti-idiotype antibodies. In other words, whether an anti-idiotype antibody can elicit antigenspecific immune responses depends on the existence of a common B-cell repertoire that recognizes antigen as well as anti-idiotype antibodies in target animals. This concept is different from the traditional view of anti-idiotype antibodies as structural antigen mimics, the so-called Ab2J3 population (18). Thus, an important criterion for selecting effective anti-idiotype antibodies is the degree of clonotypic representation in the host species. For example, when anti-idiotype antibodies are designed for human use, it is critical to utilize human antibodies as templates to generate anti-idiotype antibodies. This process should increase the chance of generating Ab3s in humans that exhibit antigen-binding potential similar to those of the template AbI antibodies. This hypothesis constitutes the basis for our use of polyclonal human neutralizing antibodies specific for the conserved CD4 site to generate anti-idiotype mAbs (3, 6). To define an Ab2 as a therapeutic vaccine candidate, the following criteria were used: (i) Ab2 should recognize human Abls that exhibit broadly neutralizing activities, and (ii) Ab2 should induce broadly neutralizing Ab3 in nonhuman primates. The similarities between 3C9-reactive human Abl and 3C9-induced monkey Ab3 can be summarized as follows: (i) both antibodies bind to gp12OsF2 and gpl20mB; (ii) they recognize an epitope around the CD4 attachment site; and (iii) they exhibit broadly neutralizing activities, although the neutralizing activities ofAb3 are limited and weaker than that of human Abl. Since certain Ab3s did not neutralize HIVmIIB strains despite their binding to gpl2OIIIB, we can speculate that the weaker or limited neutralizing activities of Ab3 may be due to a lower affinity against gpl2OIIIB. The relatively low affinity of gpl2O-specific Ab3 can be explained by the ab-
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Immunology: Kang et al.
sence of HIV antigen-primed B cells in immunologically naive monkeys. In contrast, it is anticipated that anti-idiotype antibodies will induce Ab3s of higher affinity in HIV-infected individuals who have preexisting, gp12O-primed, and affinitymatured B cells that can respond to stimulation by 3C9. Since 3C9 was not raised against monkey anti-HIV antibodies, we cannot expect a high degree of clonotypic representation in the naive B-cell repertoire of uninfected monkeys. Thus, it is not surprising to observe that the neutralizing response in the 3C9-immunized monkey is variable. Although the immunonaive monkey is not an ideal model for a HIVinfected human, we consider the data gained from the study of these animals to be very encouraging for developing therapeutic AIDS vaccines. We thank Drs. Nabil Hanna and Frank Norton for their helpful discussions, Nancy Dunlop for her technical assistance, Dr. Nancy Haigwood for providing gpl20, and Wendy Parker and Susette Fino for their assistance in the preparation of the manuscript. This study was supported in part by National Institutes of Health Grant 1R43AI31310 to C.-Y.K. and by the New York Life Insurance Company. 1. Nara, P. L., Garrity, R. D. & Goudsmit, J. (1991) FASEB J. 5, 2437-2455. 2. Ho, D. D., McKeating, J. A., Li, X. L., Moudgil, T., Daar, E. C., Sun, N.-C. & Robinson, J. E. (1991) J. Virol. 65, 489-493. 3. Chamat, S., Nara, P., Berquist, L., Whalley, A., Morrow, W. J. W., Kohler, H. & Kang, C.-Y. (1991) Modern Approaches to New Vaccines Including Prevention of AIDS: Vaccines 91 (Cold Spring Harbor Lab., Cold Spring Harbor, NY), p. 26 (abstr.). 4. Tilley, S. A., Honmen, W. J., Racho, M. E., Hilgartner, M. & Pinter, A. (1991) Res. Virol. 142, 247-259. 5. Posner, M. R., Hideshima, T., Cannon, T., Mukherjee, M., Mayer, K. H. & Byrn, R. (1991) J. Immunol. 146, 4325-4332. 6. Kang, C.-Y., Nara, P., Chamat, S., Caralli, V., Ryskamp, T., Haigwood, N., Newman, R. & Kohler, H. (1991) Proc. Natl. Acad. Sci. USA 88, 6171-6175. 7. Nara, P. L. & Fischinger, P. J. (1988) Nature (London) 332, 469-470. 8. Whalley, A. S., Nguyen, M.-L. & Morrow, W. J. W. (1991) Viral Immunol. 4, 201-213.
Proc. Natl. Acad. Sci. USA 89 (1992) 9. Daar, E. S., Li, X. L., Moudgil, T. & Ho, D. (1990) Proc. Natl. Acad. Sci. USA 87, 6574-6578. 10. Prince, M. A., Horowitz, B., Baker, L., Shulman, R. W., Ralph, H., Valinsk, J., Cundell, A., Brotman, B., Boehle, W., Rey, F., Piet, M., Reesink, A., Leslie, N., Tersmette, M., Miedema, F., Barbosa, L., Nemo, G., Nastala, C. L., Allan, J. S., Lee, D. R. & Eichberg, J. W. (1988) Proc. Natl. Acad. Sci. USA 85, 6944-6948. 11. Karpas, A., Hill, F., Youle, M., Cullen, V., Gray, J., Byron, N., Hayhoe, F. G. J., Tenant-Flowers, M., Howard, L., Gilgen, D., Oates, J. K., Hawkins, D. & Gazzard, B. (1988) Proc. Natl. Acad. Sci. USA 85, 9234-9237. 12. Emini, E. A., Schleif, W. A., Murthy, K., Eda, Y., Tokiyoshi, S., Putney, S. D., Matsushita, S., Nunberg, J. H. & Eichberg, J. W. (1991) Seventh International Conference on AIDS, Florence, TH.A.64 (Abstr.). 13. Haigwood, N. L., Nara, P. L., VanNest, G., Scandella, C. J., Eichberg, J. W. & Steimer, K. S. (1991) Modern Approaches to New Vaccines Including Prevention of AIDS: Vaccines 91 (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 51-58. 14. Morrow, W. J. W., Williams, W. M., Whalley, A. S., Ryskamp, T., Newman, R., Kang, C.-Y., Chamat, S., K6hler, H. & Kieber-Emmons, T. (1992) Immunology, in press. 15. Nisonoff, A. & Lamoyi, E. (1981) Clin. Immunol. Immunopathol. 44, 397-406. 16. Kennedy, R. C., Henkel, R. D., Pauletti, D., Allan, J. S., Lee, T. H., Essex, M. & Dreesman, G. R. (1986) Science 231, 1556-1559. 17. Kohler, H., Kaveri, S., Kieber-Emmons, T., Morrow, W. J. W., Muller, S. & Raychaudhuri, S. (1989) Methods Enzymol. 178, 3-35. 18. Jerne, N. K., Roland, J. & Cazenave, P.-A. (1982) EMBO J. 1, 243-245. 19. Schick, M. R., Dreesman, G. R. & Kennedy, R. C. (1987) J. Immunol. 138, 3419-3425. 20. Huang, J.-H., Ward, R. E. & Kohler, H. (1986) J. Immunol. 132, 770-776. 21. Kennedy, R. C., Dreesman, G. R., Butel, J. S. & Lanford, R. E. (1985) J. Exp. Med. 161, 1432-1449. 22. Kohler, H., Kieber-Emmons, T., Srinivasan, S., Kaveri, S., Morrow, W. J. W., Muller, S., Kang, C.-Y. & Raychaudhuri, S. (1989) Clin. Immunol. Immunopathol. 52, 104-116. 23. Healey, D. G., Dianda, L., Buch, D., Schroeder, K., Truneh, A., Sattentau, Q. J. & Beverly, P. C. (1991) Eur. J. Immunol. 21, 1491-1498.
