Proc. Nati. Acad. Sci. USA Vol. 88, pp. 6171-6175, July 1991
Medical Sciences
Evidence for non-V3-specific neutralizing antibodies that interfere with gpl20/CD4 binding in human immunodeficiency virus 1-infected humans (human anti-gpl20 antibody/human immunodeficiency virus 1 neutralization)
CHANG-YUIL KANG*t, PETER NARAt, SOULAIMA CHAMAT*, VINCE CARALLI*, TOM RYSKAMP*, NANCY HAIGWOOD§, ROLAND NEWMAN*, AND HEINZ KOHLER* *IDEC Pharmaceuticals Corporation, 11099 North Torrey Pines Road, La Jolla, CA 92037; tThe National Cancer Institute, Frederick, MD 21701; and §Chiron Corporation, Emeryville, CA 94608
Communicated by Maurice R. Hilleman, March 22, 1991 (received for review November 29, 1990)
Total anti-gpl20 antibodies (total anti-gpl20 ABSTRACT Abs) were purified from a pool of four human immunodeficiency virus-positive (HIV+) sera by affinity chromatography on a gp120sF2-Sepharose column and exhibited both type- and group-specific neutralizing activities. To dissect the epitope specificity of the group-specific neutrazing antibodies, CD4 attachment site-specific antibodies (CD4-site Abs) were isolated from total anti-gpl20 Abs by using a CD4-blocked gpl2OsF2Sepharose column. The CD4-site Abs exhibited group-specific neutralizing activities. Another approach to dissecting typeand group-specific neutralizing activities of total anti-gpl20 Abs was to separate the third variable region (V3)-specific antibodies (V3-region Abs) from non-V3-region-speciflc antibodies (non-V3 Abs). The results indicated that V3-region Abs exhibited type-specific neutralizing activities, whereas non-V3 Abs exhibited group-specific neutralizing activities. By comparing the neutralizing activities of V3-region Abs to those of non-V3 Abs, we concluded that V3-region Abs are more effective than non-V3 Abs in neutralizing a specific HIV isolate. Collectively, this study indicates that group-specific neutralizing anti-gpl20 antibodies are specific for the CD4 attachment site.
Although the precise role of the third variable region (V3) of gpl20 in virus infection is still in question, antibodies to this region elicited by vaccination or infection neutralize virus infectivity by interfering with a postbinding event (1-4). Furthermore, antibodies to this region exert their primary neutralizing effects on the virus strain used in immunization (5-8) and have been called "type-specific neutralizing antibodies." Under some circumstances, cross-neutralizing antibodies to the V3 domain have been observed (9). The hypervariability of the envelope region outside as well as within the V3 domain suggests that HIV mutants emerge over time that escape V3 type-specific neutralizing antibodies (10, 11). Primary, secondary, and tertiary structure of the V3region molecule are important in antibody binding and neutralization (12, 13). Since all known HIV isolates retain the ability to infect CD4+ cells despite considerable sequence variation within gpl20, it has been suggested that the CD4 attachment site on gp120 consists of conserved regions (14, 15). Antibodies binding to this CD4 attachment site are expected to block infection of a majority (if not all) of HIV isolates by interfering with the interaction with CD4+ cells. Antibodies specific against this region have been detected in the sera of HIV-infected humans (16-18). A recent report (19) described a human monoclonal antibody derived from a seropositive patient that is specific for the CD4 attachment 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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site and neutralizes multiple HIV isolates. To date no attempts have been made to our knowledge to purify these antibodies and directly correlate their activity and concentrations associated with neutralization of multiple strains of HIV-1. This study was undertaken to gain a better understanding of the humoral responses against gpl20 by defining the specificity and neutralizing function of anti-gpl20 antibodies. We have isolated and characterized various polyclonal antigpl20 antibodies from a pool of four sera of HIV-infected asymptomatic individuals. We found that (i) substantial amounts of anti-gpl20 antibodies specific for the CD4 attachment site exist in the sera, and these antibodies exert groupspecific neutralizing activities; and (ii) V3 region-specific antibodies (V3-region Abs) are more potent than non-V3region-specific antibodies (non-V3 Abs) in neutralizing a
specific isolate.
MATERIALS AND METHODS Preparation of Various Anti-gpl20 Antibodies. Total antigp120 antibodies (total anti-gp120 Abs) were purified from human sera by affinity chromatography with a HIV-1 strain SF2 gp120 (gp120sF2)-conjugated Sepharose column. Recombinant gp120sF2 was derived from engineered CHO cells. A pool of sera from HIV-infected asymptomatic individuals was inactivated by detergent (1% Nonidet P-40) and heat (1 hr at 56°C) treatments. An ammonium sulfate-precipitated immunoglobulin fraction was prepared and applied to the gp120sF2-Sepharose column. The bound total anti-gp120 Abs on the column were eluted with pH 3.0 glycine buffer. To isolate the CD4 attachment site-specific antibodies (CD4-site Abs), 10 mg of recombinant soluble CD4 (sCD4) in phosphate-buffered saline (PBS) was loaded onto the gp120sF2Sepharose column to saturate the CD4 attachment site onto the gpl20, and then 4 mg of total anti-gpl20 Abs with 10 mg of sCD4 in PBS was loaded onto the column. The flowthrough fraction was further separated on a protein G column (Pharmacia) to isolate the IgG fraction. To separate non-V3 Abs from total anti-gp120 Abs, a mixture of two peptides (8 mg of each) derived from the V3 region of gpl20sF2 (see peptides for sequence) were coupled to 10 ml of Affi-Gel 401 (Bio-Rad). Ten milligrams of total anti-gpl20 Abs in PBS was Abbreviations: total anti-gpl20 Abs, total anti-gpl20 antibodies; CD4-site Abs, CD4 attachment site-specific anti-gpl20 antibodies; V3-region Abs, V3 region-specific anti-gpl20 antibodies; non-V3Abs, non-V3-region-specific anti-gpl20 antibodies; HIV, human immunodeficiency virus; V3, the third variable region of gpl20; sCD4, soluble CD4. tTo whom reprint requests should be addressed at: IDEC Pharmaceuticals Corp., 11099 North Torrey Pines Road, #160, La Jolla, CA 92037.
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loaded on the peptide column. The flow-through, consisting of non-V3 Abs, was collected. V3-region Abs were eluted with pH 3 glycine buffer. Peptides. Peptides were synthesized on an automated peptide synthesizer (model 430A, Applied Biosystems) by the solid-phase method. The peptides were deprotected and cleaved from the support with HF/anisole or the "low-high" HF procedure (20). The amino acid sequences in single-letter code of two SF2-derived peptides for an affinity column were as follows; V3SF2: TRPNNNTRKSIYIGPGRAFHTTGRI IGDIRKAHC and CTRPNNNTRKSIYIGPGRAFHTTGRIIGDIRKAHC. The sequences of V3-region peptides from other HIV-1 strains for binding assays were as follows; strain MN, YNKRKRIHIGPGRAFYTTKNIIG; strain HIB, NNTRKSIRIQRGPGRAFVTIGKIG; strain RF, NNTRKSITKGPGRVIYATGQIIG; strain SC, NNTTRSIHIGPGRAFYATGDIIG; and strain WMJ-2, NNVRRSLSIGPGRAFRTREGN. ELISA. Plates were coated with 0.2 ml of gp120sF2 or gp12011B (SmithKline Beecham) at 0.5 Ig/ml in 0.1 M bicarbonate buffer (pH 9.8). After washing with PBS and blocking with 10%6 (vol/vol) fetal calf serum, various concentrations of anti-gp120 antibodies were added. Two hours later, biotin-coupled goat anti-human immunoglobulin and avidin-coupled peroxidase were used to detect antibody binding to the ELISA plate. To test binding activities of anti-gpl20 antibodies to V3 peptides, plates were coated overnight with 0.25 ,ug of peptide in PBS per well. The remaining steps were done as described above. Inhibition Assay on gpl20 Binding to CD4+ Cells. The assay used was a modification of the method of Schmittman et al. (16). Various concentrations of anti-gpl20 antibodies or sCD4 were added to the reaction mixtures containing 50,000 cpm of 1251-labeled gpl20 and 107 SupT-1 cells, and each mixture was incubated for 3 hr at 22°C. Cell pellets were washed and their radioactivities were measured. Percent inhibition was calculated by comparing the bound radioactivity in the presence and absence of an inhibitor. Measurement of CD4-Site Abs. Plates were coated for 18 hr with 300 ng of anti-gpl20 antibodies in PBS per well. The plates were washed and then blocked for 1 hr with 1% bovine serum albumin in PBS, and premixed 50,000 cpm of 1251. labeled gp120sF2 with various concentrations of sCD4 or without sCD4 were added to wells and incubated for 18 hr. The wells were washed, and the radioactivity of each was measured. The percent inhibition by sCD4 in each anti-gp120 antibody binding to gp120SF2 was calculated. The largest percent inhibition was considered to be the proportion of CD4-site Abs in each gpl20 antibody preparation. Neutralization Assay. A quantitative neutralization assay was used as described (21). Equivalent amounts of virus (syncytial-forming unit) were used in the assays to allow direct comparison of the various antibody fractions tested. The assays are repeatable over a virus-surviving fraction range of 1 to 0.001 within a 1:2 to 1:4 dilution of the antibody (P C 0.001).
RESULTS Isolation and Characterization of Total Anti-gpl20 Antibodies from Seropositive Sera. Sera from HIV-infected asymptomatic individuals were screened for anti-gpl20 titers. Four high-titer sera were selected and pooled. This pool was applied to a column of gpl20SF2-Sepharose. The recovery of total anti-gpl20 Abs in the sera was :300 ,ug/ml. Antibodies binding to gp120sn are expected to consist of two kinds: (i) SF2 type-specific antibodies and (it) antibodies binding to conserved regions of gpl20, such as the CD4-attachment site. Since the individuals contributing to the immunoglobulin pool might have been infected with a variety of HIV strains,
Proc. Natl. Acad. Sci. USA 88 (1991)
the flow-through fraction should bind to gpl20 derived from strains other than SF2. Indeed, we found that the flowthrough fraction bound to gpl20IIIB (Fig. 1), not to gpl20sF2, indicating the presence of IIIB-reactive anti-gpl20 antibodies in the flow-through fraction. In addition, total anti-gpl20 Abs bound to both gp12OSF2 and gp120ImB (Fig. 1), indicating the presence of a mixture of type-specific and cross-reactive anti-gpl20 antibodies. Next, we investigated the neutralizing activities of total anti-gpl20 Abs against various HIV strains. Fig. 2 shows that total anti-gpl20 Abs exhibited varied neutralizing activities against four different HIV strains, indicating that total antigpl20 Abs contains different population and amounts of typeand group-specific neutralizing antibodies. To demonstrate the presence of type-specific neutralizing antibodies in total anti-gpl20 Abs, we tested the binding of total anti-gpl20 Abs to type-specific neutralizing epitopes derived from the V3 region ofdifferent gpl20s. Table 1 shows that total anti-gpl20 Abs bind extensively to the MN epitope; less extensively to the IIIB, SC, and WMJ-2 epitopes; and weakly to the RF epitope. The highest binding activity of total anti-gpl20 Abs against the MN V3 epitope correlated with the high neutralizing activity against the MN strain. However, it was interesting to observe that the binding activity of total anti-gpl20 Abs to the RF-derived epitope was only slightly above background, despite the fact that total anti-gpl20 Abs effectively neutralized the RF strain. Isolation and Characterization of CD4-Site Abs. Since antibodies to the conserved CD4 attachment site of gpl20 are considered to be the major group-specific neutralizing epitope (15, 19), we isolated CD4-site Abs from total anti-gpl20 Abs by saturating the CD4-attachment site on gpl20 immunoabsorbent with sCD4 by applying total anti-gpl20 Abs to the immunoabsorbent in the presence of excess sCD4. The flow-through fraction of the antibodies was applied to a protein G column to separate the IgG fraction from sCD4 (Fig. 3). To ensure complete removal of the sCD4 from the IgG fraction, we analyzed this preparation by SDS/polyacrylamide gel electrophoresis and ELISA, using a monoclonal anti-CD4 antibody, and we could not detect any sCD4 (data not shown). To examine whether this IgG fraction contains CD4-site Abs, we performed competitive inhibition assays on CD4+ cells using 125I-labeled gp120s5. CD4-site Abs effectively inhibited binding of 125I-labeled gpl20sF2 to CD4 cells (Fig. 4). The IC50 of CD4-site Abs on this assay was 1/10th that of sCD4. We observed similar results with 125I-labeled gpl20OrnB (data not shown). To determine what proportion of the IgG fraction are CD4-site Abs, various anti-gp120 antibodies were coated on microtiter plates and incubated with 1-1I-labeled gpl20sF2 in the presence or absence of sCD4. Table 2 shows
4.
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-.
.
.
.
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FIG. 1. Binding of total anti-gpl20 Abs (-, A) and the flowthrough fraction (o, A) to gp12OsF2 (o, o) and to gp12OjIjB (A, A)-
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Total Anti-gpI20 Abs
gpl20 - Sepharose
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Table 1. Binding activities of different anti-gpl20 antibody preparations to peptides derived from the V3 region of various gpl2Os Binding to V3 peptides of different HIV-1 strains Anti-gpl20 antibodies MN IIIB RF SC WMJ-2 Total anti-gpl20 Abs >3 1.05 0.06 0.8 1.48 CD4-site Abs 0.15 0.01 0.01 0.03 0.01 Bindings are expressed by absorbance obtained at 5 ,g/ml of each preparation.
unbound fraction (sCD4) eluted with acidic buffer
anti-gpl 20 antibody specific against
FIG. 2. Total anti-gpl20 Abs neutralized four different strains of HIV-1. The neutralizing activity is expressed as virus-surviving fraction (Vn/Vo), where Vn is the number of syncytial-forming units of virus in the presence of total anti-gpl20 Abs, and V0 is the number of syncytial-forming units in the control well without total anti-gpl20 Abs. The results represent the average of two experiments.
that sCD4 inhibited the binding of CD4-site Abs to gpl2OsF2 by 71%, whereas only 16% of total anti-gpl20 Abs binding to gpl20sF2 was inhibited by sCD4. Thus, our purification significantly enriched CD4-site Abs; however, these antibodies still contained substantial amounts of non-CD4-attachment site-specific anti-gpl20 antibodies. To determine the specificities of the contaminating antibodies in CD4-site Abs, we tested the binding of CD4-site Abs to synthetic peptides derived from the V3 domain of different HIV-1 isolate gpl20s. Table 1 shows that CD4-site Abs bound only weakly to the MN isolate-derived epitope and not to the other isolate-derived epitopes. This finding shows that the purification for CD4-site Abs removes most of the V3-region Abs. Next, we examined whether CD4-site Abs could neutralize different HIV isolates. Indeed, CD4-site Abs were found to equivalently neutralize three different HIV strains over a similar range of antibody concentrations (Fig. 5). Compared with CD4-site Abs, total anti-gpl20 Abs exhibited much higher neutralizing activities against the MN isolate, a little higher against the RF isolate, and almost the same against the IIIB isolate. The fact that CD4-site Abs effectively neutralized three different HIV isolates at or near equivalent antibody concentrations suggests that CD4-site Abs exhibit mainly non-V3 group-specific neutralizing activities, whereas total anti-gpl20 Abs exhibit a mixture of type- and groupspecific neutralizing activities. Isolation and Characterization of Non-V3 Abs. Another approach for identifying group-specific neutralizing antibodies was to deplete V3 type-specific neutralizing antibodies from total anti-gpl20 Abs and test this preparation for groupspecific neutralizing activities. Affinity chromatography was used to separate V3- and non-V3-specific anti-gpl20 antibodies. The flow-through and bound fractions were tested for binding activities to the SF2-derived V3 peptides. Since any measurable binding of the flow-through fraction to the pep-
bound fraction
applied to protein G column
CD4 attachment site
(CD4-site Abs)
FIG. 3. Purification scheme of CD4-site Abs from total anti-gpl20 Abs.
tides was not detected (data not shown), we conclude that the
flow-through contains non-V3 Abs, while the bound fraction contains V3-region Abs only. Both bound and flow-through fractions were tested in virus
neutralization assays. Non-V3 Abs exhibited almost the same neutralizing activities against a panel of three different HIV strains (Fig. 6). However, V3-region Abs exhibited strong neutralization activities against the MN isolate, minimal activities against IIIB, and no activity against RF. This result indicates that the majority of neutralizing activity of total anti-gpl20 Abs to IIIB and RF isolates (Fig. 6 Left and Center) are derived from group-specific neutralizing activities ofthe non-V3 Abs fraction. Most of the neutralizing activity of total anti-gpl20 Abs to the MN isolates (Fig. 6 Right) was derived from antibodies recognizing the type-specific V3 region. By comparing neutralizing activities of V3-region Abs and non-V3 Abs on MN isolate (Fig. 6 Right), we concluded that V3-region Abs are more effective than non-V3 Abs in neutralizing a specific isolate. extent of
DISCUSSION In this study, we have dissected the neutralizing activities of various polyclonal anti-gp120 antibodies present in a pool of serum from four HIV-infected individuals. The major find80
-
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Inhibitor, nM FIG. 4. Inhibition of gpl20 binding to CD4 by CD4-site Abs, sCD4, and normal IgG. Supt-1 cells (107) were incubated for 2 hr with 50,000 cpm of "WI-labeled gp120sF2 in the presence or absence of inhibitors. The percent inhibition was calculated. The results represent the average of two
experiments.
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Table 2. Proportion of CD4-attachment site-specific antibodies in different anti-gp120 antibody preparations Anti-gp120 Proportion* antibodies (as %) Total anti-gp120 Abs 16 CD4-site Abs 71 Non-V3 Abs 32 V3-region Abs 0 *The percent inhibition by sCD4 in each anti-gpl20 antibody binding to gp120 was calculated. The largest percent inhibition was considered to be the proportion of CD4 attachment site-specific antigp120 antibodies.
ings of this study are: (i) total anti-gpl20 Abs exert type- and group-specific neutralizing activities; (it) the group-specific neutralizing activity is found in CD4-site Abs; (iii) if other group-specific neutralizing antibodies rather than CD4-site Abs exist, they are not specific against a simple linear epitope of the V3 domain; and (iv) V3-region Abs are more effective than non-V3 Abs in neutralizing a specific HIV isolate. HIV+ sera exert group-specific neutralizing activities (2224). Recent reports (25, 26) also demonstrated that the group-specific neutralizing activities disappeared after absorption of the sera on gpl60. The group-specific neutralizing activities of total anti-gpl20 Abs described here clearly indicate that this activity is contributed, at least in part, by anti-gpl20 antibodies. The highest neutralizing activities of total anti-gpl20 Abs against the MN isolate agreed with the highest binding activities of total anti-gp120 Abs against the V3 type-specific neutralizing epitope derived from the MN isolate. This indicated that total anti-gpl20 Abs contained type-specific neutralizing antibodies that were more specific against the MN isolate than the other HIV isolates. Since the SF2 and MN isolates belong to the same group in terms of serological definition of the V3-specific antibodies (12), the purification of total anti-gpl20 Abs on gp120sF2-Sepharose preferentially selected type-specific neutralizing antibodies against the SF2 and MN isolates. Although antibodies specific against the CD4 attachment site on gpl20 exist in the sera ofHIV-infected individuals (16, 17) and a CD4-site-specific human monoclonal antibody (19) has been described, it is still uncertain to what extent these antibodies contribute to host defense against viral dissemination. Purified CD4-site Abs, indeed, neutralized almost equally three HIV strains. Despite the fact that the CD4-site Abs fraction did not contain type-specific neutralizing antibodies against either RF or IIIB isolates (Table 1), this fraction still neutralized HIVRF and HIVIIIB (Fig. 5 Left and
Center). From this observation, we conclude that the groupspecific neutralizing activity in total anti-gpl20 Abs can be accounted for to a large extent by the CD4-site Abs fraction. Several reports have indicated that antibodies against the V3-region on gpl20 exert type-specific neutralizing activities (6-8). Previous studies (27) using denatured and native gpl2O5s antigens have shown that only type-specific neutralizing antibodies are removed from human serum by the denatured antigen, while group-specific neutralizing antibodies can bind to the native antigen used in the studies described here. We separated the V3-region- and non-V3-regionspecific antibodies from total anti-gpl20 Abs to distinguish their neutralizing activities against various HIV isolates. The results described here clearly demonstrate that the V3-region Abs exert type-specific neutralizing activities, whereas non-V3 Abs exert group-specific neutralizing activities. Onethird of non-V3 Abs were found to be inhibited by sCD4, demonstrating that a significant portion of these antibodies are directed against the CD4 attachment site. We do not know the specificity of the remaining two-thirds of anti-gpl20 antibodies in the non-V3 Abs and whether these antibodies have neutralizing activity. This implies that these two-thirds of non-V3 Abs may not have neutralizing activity. If this is the case, the CD4-site Abs, being one-third of non-V3 Abs, would represent the entire neutralizing activity of non-V3 Abs. However, this result cannot eliminate the possibility that the neutralizing activities of non-V3 Abs, at least in part, are derived from antibodies with unknown specificities or from certain antibodies that recognize conformational epitopes of the V3-region (12, 26). By comparing the neutralizing activities of V3-region Abs and non-V3 Abs in HIVMN (Fig. 6 Right), we concluded that V3-region Abs were more effective than non-V3 Abs in neutralizing a specific isolate. However, our interpretation and conclusion may be influenced by additional factors such as purity and affinity of our antibody preparations and the virus isolates used in the neutralization assays. The recovery of the V3-region Abs from total anti-gp120 Abs by using an affinity column was in the range of 15%. Inhibition of binding of total anti-gpl20 Abs to gpl20 by sCD4 reached 16%o, indicating that a fraction of anti-gp120 antibodies similar to the anti-V3 Abs is directed to the CD4 attachment site. Since total anti-gpl20 Abs were purified by using a pool of four sera, the estimated value may be representative for the sera of HIV-infected asymptomatic individuals. Based on this observation, it is of interest to consider the contribution of CD4-site Abs and V3-region Abs in preventing viral infection in vivo. V3-region Abs have been
RF
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1
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FIG. 5. Neutralizing activities oftotal anti-gpl20 Abs and CD4-site Abs against three different HIV-1 isolates; HIVIIIB (Left), HIVRF (Center), and HIVMN (Right). The neutralizing activity is expressed in the same virus-surviving fraction terms as in Fig. 2. The results represent the average of two experiments.
Medical Sciences: Kang et aL
RF
Proc. Natl. Acad. Sci. USA 88 (1991)
MN
IIIB 1.0 -
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V3-reglon Abs
*
Nnn-V3 Abs
v Toa g AntlI pL2Ab
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Ab, Conc. Atg/ml FIG. 6. Neutralizing activities of total anti-gpl20 Abs, non-V3 Abs, and V3-region Abs against three different HIV-1 isolates: HIVRF (Left), HIVIIIB (Center), and HIVMN (Right). The neutralizing activity is expressed in the same virus-surviving fraction terms as in Fig. 2. The results represent the average of two experiments.
shown (11) to play an important role in the beginning stage of infection via their early and rapid induction and high neutralizing efficacy. However, the role of V3-region Abs might be diminished with time since V3-region Abs no longer effectively neutralize newly emerging mutated isolates (4, 11). In such cases, CD4-site Abs might neutralize the V3 escape mutants, since the CD4 attachment site on gp120 is expected to be a highly conserved region (28). In this regard it should be interesting to determine if primary virus isolates as well as escape mutants are as effectively neutralized by the different anti-gp120 antibody preparations as are established laboratory HIV-1 strains. The authors thank Drs. Frank Norton, John Morrow, and Nabil Hanna for their helpful discussions; Agnes Chen, Greg Warner, and Nancy Dunlop for their technical assistance; and Wendy Parker for her assistance in the preparation of the manuscript. This study was supported in part by Grant IR43A129770-01 from the National Institute of Allergy and Infectious Diseases. 1. Nara, P. L. (1988) in Colloque Des Cent Gardes, eds. Girard, M. & Valette, L. (Pasteur Vaccins, Paris), pp. 138-150. 2. Skinner, M. A., Langlois, A. J., McDanal, C. B., McDougal, J. S., Bolognesi, D. P. & Matthews, T. J. (1988) J. Virol. 62, 3. 4. 5.
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21.
Redfield, R. R., Burke, D. S., Gallo, R. C. & Wong-Staal, F. (1988) Science 241, 357-359. Nara, P. L., Smit, L., Dunlop, N., Hatch, W., Merges, M., Waters, J., Kelliher, R., Gallo, C., Fischinger, P. J. & Goudsmit, J. (1990) J. Virol. 64, 3779-3791. Goudsmit, J., Kuibean, C. L. & Nara, P. L. (1989) AIDS 3, Suppl. 1, S119-S123. Clements, G. J., Price-Jones, M. J., Stephens, P. E., Sutton, C., Schultz, T. F., Clapham, P., McKeating, J. A., McClure, M. O., Thomson, S., March, M., Kay, I., Weiss, R. A. & Moore, J. P. (1991) AIDS Res. Hum. Retroviruses 7, 3-16. Kowalski, M., Potz, J., Basiripour, L., Dorfman, T., Goh, W. C., Terwilliger, E., Dayton, A., Rosen, C., Haseltine, W. & Sordroski, J. (1987) Science 237, 1351-1355. Syu, W.-J., Huang, J.-H., Essex, M. & Lee, T.-H. (1990) Proc. Natl. Acad. Sci. USA 87, 3695-3699. Schmittan, S. M., Lane, H. C., Roth, J., Burrows, A., Folks, T. M., Kehal, J. H., Koenig, S., Berman, P. & Fauci, A. S. (1988) J. Immunol. 141, 4181-4186. Moore, J. P. (1990) AIDS 4, 297-305. Back, N. K. T., Thiriart, C., Delers, A., Ramautarsing, C., Bruck, C. & Goudsmit, J. (1990) J. Med. Virol. 31, 200-208. Ho, D. D., McKeating, J. A., Li, X. L., Moudgil, T., Daar, E. C., Slun, N.-C. & Robinson, J. E. (1991) J. Virol. 65, 489-493. Tam, J., Heath, W. F. & Merrifield, R. (1983) J. Am. Chem. Soc. 21, 6442-6450. Nara, P. L. & Fischinger, P. J. (1988) Nature (London) 332,
469-470. 22. Weiss, R. A., Clapham, P. R., Cheingsong-Popov, R., Dalgleish, A. G., Carne, A. C., Weller, I. V. D. & Tedder, R. S. (1985) Nature (London) 316, 69-72. 23. Robert-Guroff, M., Brown, M. & Gallo, R. C. (1985) Nature (London) 316, 72-74. 24. Ho, D. D., Sarngadharan, M. G., Hirsch, M. S., Schooley, R. T., Rota, T. R., Kennedy, R. C., Chanh, T. C. & Sato, V. L. (1987) J. Virol. 61, 2024-2028. 25. Berkower, I., Smith, G. E., Giri, C. & Murphy, D. (1989) J. Exp. Med. 170, 1681-1695. 26. Profy, A. T., Salinas, D. A., Eckler, L. I., Dunlop, N. M., Nara, P. L. & Putney, S. D. (1990) J. Immunol. 144, 46414647. 27. Haigwood, N. L., Barker, C. B., Higgins, K. W., Skiles, P. V., Moore, G. K., Mann, K. A., Lee, D. R., Eichberg, J. W. & Steimer, K. S. (1990) in Modern Approaches to New Vaccines Including Prevention of AIDS: Vaccines 90 (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 313-320. 28. Johnson, P. R., Hamm, T. E. & Hirsch, V. M. (1990) in Modern Approaches to New Vaccines Including Prevention of AIDS: Vaccine 90 (Cold Spring Harbor Lab., Cold Spring Harbor, NY), p. 85.
Medical Sciences
Evidence for non-V3-specific neutralizing antibodies that interfere with gpl20/CD4 binding in human immunodeficiency virus 1-infected humans (human anti-gpl20 antibody/human immunodeficiency virus 1 neutralization)
CHANG-YUIL KANG*t, PETER NARAt, SOULAIMA CHAMAT*, VINCE CARALLI*, TOM RYSKAMP*, NANCY HAIGWOOD§, ROLAND NEWMAN*, AND HEINZ KOHLER* *IDEC Pharmaceuticals Corporation, 11099 North Torrey Pines Road, La Jolla, CA 92037; tThe National Cancer Institute, Frederick, MD 21701; and §Chiron Corporation, Emeryville, CA 94608
Communicated by Maurice R. Hilleman, March 22, 1991 (received for review November 29, 1990)
Total anti-gpl20 antibodies (total anti-gpl20 ABSTRACT Abs) were purified from a pool of four human immunodeficiency virus-positive (HIV+) sera by affinity chromatography on a gp120sF2-Sepharose column and exhibited both type- and group-specific neutralizing activities. To dissect the epitope specificity of the group-specific neutrazing antibodies, CD4 attachment site-specific antibodies (CD4-site Abs) were isolated from total anti-gpl20 Abs by using a CD4-blocked gpl2OsF2Sepharose column. The CD4-site Abs exhibited group-specific neutralizing activities. Another approach to dissecting typeand group-specific neutralizing activities of total anti-gpl20 Abs was to separate the third variable region (V3)-specific antibodies (V3-region Abs) from non-V3-region-speciflc antibodies (non-V3 Abs). The results indicated that V3-region Abs exhibited type-specific neutralizing activities, whereas non-V3 Abs exhibited group-specific neutralizing activities. By comparing the neutralizing activities of V3-region Abs to those of non-V3 Abs, we concluded that V3-region Abs are more effective than non-V3 Abs in neutralizing a specific HIV isolate. Collectively, this study indicates that group-specific neutralizing anti-gpl20 antibodies are specific for the CD4 attachment site.
Although the precise role of the third variable region (V3) of gpl20 in virus infection is still in question, antibodies to this region elicited by vaccination or infection neutralize virus infectivity by interfering with a postbinding event (1-4). Furthermore, antibodies to this region exert their primary neutralizing effects on the virus strain used in immunization (5-8) and have been called "type-specific neutralizing antibodies." Under some circumstances, cross-neutralizing antibodies to the V3 domain have been observed (9). The hypervariability of the envelope region outside as well as within the V3 domain suggests that HIV mutants emerge over time that escape V3 type-specific neutralizing antibodies (10, 11). Primary, secondary, and tertiary structure of the V3region molecule are important in antibody binding and neutralization (12, 13). Since all known HIV isolates retain the ability to infect CD4+ cells despite considerable sequence variation within gpl20, it has been suggested that the CD4 attachment site on gp120 consists of conserved regions (14, 15). Antibodies binding to this CD4 attachment site are expected to block infection of a majority (if not all) of HIV isolates by interfering with the interaction with CD4+ cells. Antibodies specific against this region have been detected in the sera of HIV-infected humans (16-18). A recent report (19) described a human monoclonal antibody derived from a seropositive patient that is specific for the CD4 attachment 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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site and neutralizes multiple HIV isolates. To date no attempts have been made to our knowledge to purify these antibodies and directly correlate their activity and concentrations associated with neutralization of multiple strains of HIV-1. This study was undertaken to gain a better understanding of the humoral responses against gpl20 by defining the specificity and neutralizing function of anti-gpl20 antibodies. We have isolated and characterized various polyclonal antigpl20 antibodies from a pool of four sera of HIV-infected asymptomatic individuals. We found that (i) substantial amounts of anti-gpl20 antibodies specific for the CD4 attachment site exist in the sera, and these antibodies exert groupspecific neutralizing activities; and (ii) V3 region-specific antibodies (V3-region Abs) are more potent than non-V3region-specific antibodies (non-V3 Abs) in neutralizing a
specific isolate.
MATERIALS AND METHODS Preparation of Various Anti-gpl20 Antibodies. Total antigp120 antibodies (total anti-gp120 Abs) were purified from human sera by affinity chromatography with a HIV-1 strain SF2 gp120 (gp120sF2)-conjugated Sepharose column. Recombinant gp120sF2 was derived from engineered CHO cells. A pool of sera from HIV-infected asymptomatic individuals was inactivated by detergent (1% Nonidet P-40) and heat (1 hr at 56°C) treatments. An ammonium sulfate-precipitated immunoglobulin fraction was prepared and applied to the gp120sF2-Sepharose column. The bound total anti-gp120 Abs on the column were eluted with pH 3.0 glycine buffer. To isolate the CD4 attachment site-specific antibodies (CD4-site Abs), 10 mg of recombinant soluble CD4 (sCD4) in phosphate-buffered saline (PBS) was loaded onto the gp120sF2Sepharose column to saturate the CD4 attachment site onto the gpl20, and then 4 mg of total anti-gpl20 Abs with 10 mg of sCD4 in PBS was loaded onto the column. The flowthrough fraction was further separated on a protein G column (Pharmacia) to isolate the IgG fraction. To separate non-V3 Abs from total anti-gp120 Abs, a mixture of two peptides (8 mg of each) derived from the V3 region of gpl20sF2 (see peptides for sequence) were coupled to 10 ml of Affi-Gel 401 (Bio-Rad). Ten milligrams of total anti-gpl20 Abs in PBS was Abbreviations: total anti-gpl20 Abs, total anti-gpl20 antibodies; CD4-site Abs, CD4 attachment site-specific anti-gpl20 antibodies; V3-region Abs, V3 region-specific anti-gpl20 antibodies; non-V3Abs, non-V3-region-specific anti-gpl20 antibodies; HIV, human immunodeficiency virus; V3, the third variable region of gpl20; sCD4, soluble CD4. tTo whom reprint requests should be addressed at: IDEC Pharmaceuticals Corp., 11099 North Torrey Pines Road, #160, La Jolla, CA 92037.
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loaded on the peptide column. The flow-through, consisting of non-V3 Abs, was collected. V3-region Abs were eluted with pH 3 glycine buffer. Peptides. Peptides were synthesized on an automated peptide synthesizer (model 430A, Applied Biosystems) by the solid-phase method. The peptides were deprotected and cleaved from the support with HF/anisole or the "low-high" HF procedure (20). The amino acid sequences in single-letter code of two SF2-derived peptides for an affinity column were as follows; V3SF2: TRPNNNTRKSIYIGPGRAFHTTGRI IGDIRKAHC and CTRPNNNTRKSIYIGPGRAFHTTGRIIGDIRKAHC. The sequences of V3-region peptides from other HIV-1 strains for binding assays were as follows; strain MN, YNKRKRIHIGPGRAFYTTKNIIG; strain HIB, NNTRKSIRIQRGPGRAFVTIGKIG; strain RF, NNTRKSITKGPGRVIYATGQIIG; strain SC, NNTTRSIHIGPGRAFYATGDIIG; and strain WMJ-2, NNVRRSLSIGPGRAFRTREGN. ELISA. Plates were coated with 0.2 ml of gp120sF2 or gp12011B (SmithKline Beecham) at 0.5 Ig/ml in 0.1 M bicarbonate buffer (pH 9.8). After washing with PBS and blocking with 10%6 (vol/vol) fetal calf serum, various concentrations of anti-gp120 antibodies were added. Two hours later, biotin-coupled goat anti-human immunoglobulin and avidin-coupled peroxidase were used to detect antibody binding to the ELISA plate. To test binding activities of anti-gpl20 antibodies to V3 peptides, plates were coated overnight with 0.25 ,ug of peptide in PBS per well. The remaining steps were done as described above. Inhibition Assay on gpl20 Binding to CD4+ Cells. The assay used was a modification of the method of Schmittman et al. (16). Various concentrations of anti-gpl20 antibodies or sCD4 were added to the reaction mixtures containing 50,000 cpm of 1251-labeled gpl20 and 107 SupT-1 cells, and each mixture was incubated for 3 hr at 22°C. Cell pellets were washed and their radioactivities were measured. Percent inhibition was calculated by comparing the bound radioactivity in the presence and absence of an inhibitor. Measurement of CD4-Site Abs. Plates were coated for 18 hr with 300 ng of anti-gpl20 antibodies in PBS per well. The plates were washed and then blocked for 1 hr with 1% bovine serum albumin in PBS, and premixed 50,000 cpm of 1251. labeled gp120sF2 with various concentrations of sCD4 or without sCD4 were added to wells and incubated for 18 hr. The wells were washed, and the radioactivity of each was measured. The percent inhibition by sCD4 in each anti-gp120 antibody binding to gp120SF2 was calculated. The largest percent inhibition was considered to be the proportion of CD4-site Abs in each gpl20 antibody preparation. Neutralization Assay. A quantitative neutralization assay was used as described (21). Equivalent amounts of virus (syncytial-forming unit) were used in the assays to allow direct comparison of the various antibody fractions tested. The assays are repeatable over a virus-surviving fraction range of 1 to 0.001 within a 1:2 to 1:4 dilution of the antibody (P C 0.001).
RESULTS Isolation and Characterization of Total Anti-gpl20 Antibodies from Seropositive Sera. Sera from HIV-infected asymptomatic individuals were screened for anti-gpl20 titers. Four high-titer sera were selected and pooled. This pool was applied to a column of gpl20SF2-Sepharose. The recovery of total anti-gpl20 Abs in the sera was :300 ,ug/ml. Antibodies binding to gp120sn are expected to consist of two kinds: (i) SF2 type-specific antibodies and (it) antibodies binding to conserved regions of gpl20, such as the CD4-attachment site. Since the individuals contributing to the immunoglobulin pool might have been infected with a variety of HIV strains,
Proc. Natl. Acad. Sci. USA 88 (1991)
the flow-through fraction should bind to gpl20 derived from strains other than SF2. Indeed, we found that the flowthrough fraction bound to gpl20IIIB (Fig. 1), not to gpl20sF2, indicating the presence of IIIB-reactive anti-gpl20 antibodies in the flow-through fraction. In addition, total anti-gpl20 Abs bound to both gp12OSF2 and gp120ImB (Fig. 1), indicating the presence of a mixture of type-specific and cross-reactive anti-gpl20 antibodies. Next, we investigated the neutralizing activities of total anti-gpl20 Abs against various HIV strains. Fig. 2 shows that total anti-gpl20 Abs exhibited varied neutralizing activities against four different HIV strains, indicating that total antigpl20 Abs contains different population and amounts of typeand group-specific neutralizing antibodies. To demonstrate the presence of type-specific neutralizing antibodies in total anti-gpl20 Abs, we tested the binding of total anti-gpl20 Abs to type-specific neutralizing epitopes derived from the V3 region ofdifferent gpl20s. Table 1 shows that total anti-gpl20 Abs bind extensively to the MN epitope; less extensively to the IIIB, SC, and WMJ-2 epitopes; and weakly to the RF epitope. The highest binding activity of total anti-gpl20 Abs against the MN V3 epitope correlated with the high neutralizing activity against the MN strain. However, it was interesting to observe that the binding activity of total anti-gpl20 Abs to the RF-derived epitope was only slightly above background, despite the fact that total anti-gpl20 Abs effectively neutralized the RF strain. Isolation and Characterization of CD4-Site Abs. Since antibodies to the conserved CD4 attachment site of gpl20 are considered to be the major group-specific neutralizing epitope (15, 19), we isolated CD4-site Abs from total anti-gpl20 Abs by saturating the CD4-attachment site on gpl20 immunoabsorbent with sCD4 by applying total anti-gpl20 Abs to the immunoabsorbent in the presence of excess sCD4. The flow-through fraction of the antibodies was applied to a protein G column to separate the IgG fraction from sCD4 (Fig. 3). To ensure complete removal of the sCD4 from the IgG fraction, we analyzed this preparation by SDS/polyacrylamide gel electrophoresis and ELISA, using a monoclonal anti-CD4 antibody, and we could not detect any sCD4 (data not shown). To examine whether this IgG fraction contains CD4-site Abs, we performed competitive inhibition assays on CD4+ cells using 125I-labeled gp120s5. CD4-site Abs effectively inhibited binding of 125I-labeled gpl20sF2 to CD4 cells (Fig. 4). The IC50 of CD4-site Abs on this assay was 1/10th that of sCD4. We observed similar results with 125I-labeled gpl20OrnB (data not shown). To determine what proportion of the IgG fraction are CD4-site Abs, various anti-gp120 antibodies were coated on microtiter plates and incubated with 1-1I-labeled gpl20sF2 in the presence or absence of sCD4. Table 2 shows
4.
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r:
I
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1*
,* I
I
I
I
Il
±vII ...
0.0001
...
.1
0.001
-.
.
.
.
. . . ......
0.1 0.01 Ab, gg/ml
...
1
10
FIG. 1. Binding of total anti-gpl20 Abs (-, A) and the flowthrough fraction (o, A) to gp12OsF2 (o, o) and to gp12OjIjB (A, A)-
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Kang et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
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Total Anti-gpI20 Abs
gpl20 - Sepharose
Y o
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cc
Ia
blocked by sCD4
0.4 unbound fraction
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0.2 bound fraction
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1
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Table 1. Binding activities of different anti-gpl20 antibody preparations to peptides derived from the V3 region of various gpl2Os Binding to V3 peptides of different HIV-1 strains Anti-gpl20 antibodies MN IIIB RF SC WMJ-2 Total anti-gpl20 Abs >3 1.05 0.06 0.8 1.48 CD4-site Abs 0.15 0.01 0.01 0.03 0.01 Bindings are expressed by absorbance obtained at 5 ,g/ml of each preparation.
unbound fraction (sCD4) eluted with acidic buffer
anti-gpl 20 antibody specific against
FIG. 2. Total anti-gpl20 Abs neutralized four different strains of HIV-1. The neutralizing activity is expressed as virus-surviving fraction (Vn/Vo), where Vn is the number of syncytial-forming units of virus in the presence of total anti-gpl20 Abs, and V0 is the number of syncytial-forming units in the control well without total anti-gpl20 Abs. The results represent the average of two experiments.
that sCD4 inhibited the binding of CD4-site Abs to gpl2OsF2 by 71%, whereas only 16% of total anti-gpl20 Abs binding to gpl20sF2 was inhibited by sCD4. Thus, our purification significantly enriched CD4-site Abs; however, these antibodies still contained substantial amounts of non-CD4-attachment site-specific anti-gpl20 antibodies. To determine the specificities of the contaminating antibodies in CD4-site Abs, we tested the binding of CD4-site Abs to synthetic peptides derived from the V3 domain of different HIV-1 isolate gpl20s. Table 1 shows that CD4-site Abs bound only weakly to the MN isolate-derived epitope and not to the other isolate-derived epitopes. This finding shows that the purification for CD4-site Abs removes most of the V3-region Abs. Next, we examined whether CD4-site Abs could neutralize different HIV isolates. Indeed, CD4-site Abs were found to equivalently neutralize three different HIV strains over a similar range of antibody concentrations (Fig. 5). Compared with CD4-site Abs, total anti-gpl20 Abs exhibited much higher neutralizing activities against the MN isolate, a little higher against the RF isolate, and almost the same against the IIIB isolate. The fact that CD4-site Abs effectively neutralized three different HIV isolates at or near equivalent antibody concentrations suggests that CD4-site Abs exhibit mainly non-V3 group-specific neutralizing activities, whereas total anti-gpl20 Abs exhibit a mixture of type- and groupspecific neutralizing activities. Isolation and Characterization of Non-V3 Abs. Another approach for identifying group-specific neutralizing antibodies was to deplete V3 type-specific neutralizing antibodies from total anti-gpl20 Abs and test this preparation for groupspecific neutralizing activities. Affinity chromatography was used to separate V3- and non-V3-specific anti-gpl20 antibodies. The flow-through and bound fractions were tested for binding activities to the SF2-derived V3 peptides. Since any measurable binding of the flow-through fraction to the pep-
bound fraction
applied to protein G column
CD4 attachment site
(CD4-site Abs)
FIG. 3. Purification scheme of CD4-site Abs from total anti-gpl20 Abs.
tides was not detected (data not shown), we conclude that the
flow-through contains non-V3 Abs, while the bound fraction contains V3-region Abs only. Both bound and flow-through fractions were tested in virus
neutralization assays. Non-V3 Abs exhibited almost the same neutralizing activities against a panel of three different HIV strains (Fig. 6). However, V3-region Abs exhibited strong neutralization activities against the MN isolate, minimal activities against IIIB, and no activity against RF. This result indicates that the majority of neutralizing activity of total anti-gpl20 Abs to IIIB and RF isolates (Fig. 6 Left and Center) are derived from group-specific neutralizing activities ofthe non-V3 Abs fraction. Most of the neutralizing activity of total anti-gpl20 Abs to the MN isolates (Fig. 6 Right) was derived from antibodies recognizing the type-specific V3 region. By comparing neutralizing activities of V3-region Abs and non-V3 Abs on MN isolate (Fig. 6 Right), we concluded that V3-region Abs are more effective than non-V3 Abs in neutralizing a specific isolate. extent of
DISCUSSION In this study, we have dissected the neutralizing activities of various polyclonal anti-gp120 antibodies present in a pool of serum from four HIV-infected individuals. The major find80
-
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-*
sCD4
*
Normal IgG
60 0
9 40 20
0~
10-1
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10'
102
103~
Inhibitor, nM FIG. 4. Inhibition of gpl20 binding to CD4 by CD4-site Abs, sCD4, and normal IgG. Supt-1 cells (107) were incubated for 2 hr with 50,000 cpm of "WI-labeled gp120sF2 in the presence or absence of inhibitors. The percent inhibition was calculated. The results represent the average of two
experiments.
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Table 2. Proportion of CD4-attachment site-specific antibodies in different anti-gp120 antibody preparations Anti-gp120 Proportion* antibodies (as %) Total anti-gp120 Abs 16 CD4-site Abs 71 Non-V3 Abs 32 V3-region Abs 0 *The percent inhibition by sCD4 in each anti-gpl20 antibody binding to gp120 was calculated. The largest percent inhibition was considered to be the proportion of CD4 attachment site-specific antigp120 antibodies.
ings of this study are: (i) total anti-gpl20 Abs exert type- and group-specific neutralizing activities; (it) the group-specific neutralizing activity is found in CD4-site Abs; (iii) if other group-specific neutralizing antibodies rather than CD4-site Abs exist, they are not specific against a simple linear epitope of the V3 domain; and (iv) V3-region Abs are more effective than non-V3 Abs in neutralizing a specific HIV isolate. HIV+ sera exert group-specific neutralizing activities (2224). Recent reports (25, 26) also demonstrated that the group-specific neutralizing activities disappeared after absorption of the sera on gpl60. The group-specific neutralizing activities of total anti-gpl20 Abs described here clearly indicate that this activity is contributed, at least in part, by anti-gpl20 antibodies. The highest neutralizing activities of total anti-gpl20 Abs against the MN isolate agreed with the highest binding activities of total anti-gp120 Abs against the V3 type-specific neutralizing epitope derived from the MN isolate. This indicated that total anti-gpl20 Abs contained type-specific neutralizing antibodies that were more specific against the MN isolate than the other HIV isolates. Since the SF2 and MN isolates belong to the same group in terms of serological definition of the V3-specific antibodies (12), the purification of total anti-gpl20 Abs on gp120sF2-Sepharose preferentially selected type-specific neutralizing antibodies against the SF2 and MN isolates. Although antibodies specific against the CD4 attachment site on gpl20 exist in the sera ofHIV-infected individuals (16, 17) and a CD4-site-specific human monoclonal antibody (19) has been described, it is still uncertain to what extent these antibodies contribute to host defense against viral dissemination. Purified CD4-site Abs, indeed, neutralized almost equally three HIV strains. Despite the fact that the CD4-site Abs fraction did not contain type-specific neutralizing antibodies against either RF or IIIB isolates (Table 1), this fraction still neutralized HIVRF and HIVIIIB (Fig. 5 Left and
Center). From this observation, we conclude that the groupspecific neutralizing activity in total anti-gpl20 Abs can be accounted for to a large extent by the CD4-site Abs fraction. Several reports have indicated that antibodies against the V3-region on gpl20 exert type-specific neutralizing activities (6-8). Previous studies (27) using denatured and native gpl2O5s antigens have shown that only type-specific neutralizing antibodies are removed from human serum by the denatured antigen, while group-specific neutralizing antibodies can bind to the native antigen used in the studies described here. We separated the V3-region- and non-V3-regionspecific antibodies from total anti-gpl20 Abs to distinguish their neutralizing activities against various HIV isolates. The results described here clearly demonstrate that the V3-region Abs exert type-specific neutralizing activities, whereas non-V3 Abs exert group-specific neutralizing activities. Onethird of non-V3 Abs were found to be inhibited by sCD4, demonstrating that a significant portion of these antibodies are directed against the CD4 attachment site. We do not know the specificity of the remaining two-thirds of anti-gpl20 antibodies in the non-V3 Abs and whether these antibodies have neutralizing activity. This implies that these two-thirds of non-V3 Abs may not have neutralizing activity. If this is the case, the CD4-site Abs, being one-third of non-V3 Abs, would represent the entire neutralizing activity of non-V3 Abs. However, this result cannot eliminate the possibility that the neutralizing activities of non-V3 Abs, at least in part, are derived from antibodies with unknown specificities or from certain antibodies that recognize conformational epitopes of the V3-region (12, 26). By comparing the neutralizing activities of V3-region Abs and non-V3 Abs in HIVMN (Fig. 6 Right), we concluded that V3-region Abs were more effective than non-V3 Abs in neutralizing a specific isolate. However, our interpretation and conclusion may be influenced by additional factors such as purity and affinity of our antibody preparations and the virus isolates used in the neutralization assays. The recovery of the V3-region Abs from total anti-gp120 Abs by using an affinity column was in the range of 15%. Inhibition of binding of total anti-gpl20 Abs to gpl20 by sCD4 reached 16%o, indicating that a fraction of anti-gp120 antibodies similar to the anti-V3 Abs is directed to the CD4 attachment site. Since total anti-gpl20 Abs were purified by using a pool of four sera, the estimated value may be representative for the sera of HIV-infected asymptomatic individuals. Based on this observation, it is of interest to consider the contribution of CD4-site Abs and V3-region Abs in preventing viral infection in vivo. V3-region Abs have been
RF
ItB
MN
1.0 -
9
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Total Antl-gp 1 20 Abs CD4-site Abs
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0.2-0.
0.1
1
10
100
0.1
1
10
100
0.1
1
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100
Ab, ug/ml
FIG. 5. Neutralizing activities oftotal anti-gpl20 Abs and CD4-site Abs against three different HIV-1 isolates; HIVIIIB (Left), HIVRF (Center), and HIVMN (Right). The neutralizing activity is expressed in the same virus-surviving fraction terms as in Fig. 2. The results represent the average of two experiments.
Medical Sciences: Kang et aL
RF
Proc. Natl. Acad. Sci. USA 88 (1991)
MN
IIIB 1.0 -
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V3-reglon Abs
*
Nnn-V3 Abs
v Toa g AntlI pL2Ab
Total Anti-gp 120 Abs 0.8-
0.6-
o
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1
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1100
0.0 0.1
1
10
100
Ab, Conc. Atg/ml FIG. 6. Neutralizing activities of total anti-gpl20 Abs, non-V3 Abs, and V3-region Abs against three different HIV-1 isolates: HIVRF (Left), HIVIIIB (Center), and HIVMN (Right). The neutralizing activity is expressed in the same virus-surviving fraction terms as in Fig. 2. The results represent the average of two experiments.
shown (11) to play an important role in the beginning stage of infection via their early and rapid induction and high neutralizing efficacy. However, the role of V3-region Abs might be diminished with time since V3-region Abs no longer effectively neutralize newly emerging mutated isolates (4, 11). In such cases, CD4-site Abs might neutralize the V3 escape mutants, since the CD4 attachment site on gp120 is expected to be a highly conserved region (28). In this regard it should be interesting to determine if primary virus isolates as well as escape mutants are as effectively neutralized by the different anti-gp120 antibody preparations as are established laboratory HIV-1 strains. The authors thank Drs. Frank Norton, John Morrow, and Nabil Hanna for their helpful discussions; Agnes Chen, Greg Warner, and Nancy Dunlop for their technical assistance; and Wendy Parker for her assistance in the preparation of the manuscript. This study was supported in part by Grant IR43A129770-01 from the National Institute of Allergy and Infectious Diseases. 1. Nara, P. L. (1988) in Colloque Des Cent Gardes, eds. Girard, M. & Valette, L. (Pasteur Vaccins, Paris), pp. 138-150. 2. Skinner, M. A., Langlois, A. J., McDanal, C. B., McDougal, J. S., Bolognesi, D. P. & Matthews, T. J. (1988) J. Virol. 62, 3. 4. 5.
6. 7. 8.
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