Proc. Nati. Acad. Sci. USA Vol. 88, pp. 7056-7060, August 1991 Medical Sciences

Resistance of primary isolates of human immunodeficiency virus type 1 to soluble CD4 is independent of CD4-rgpl2O binding affinity (AIDS/antiviral therapy/envelope glycoprotein gpl2O/receptor)

Avi ASHKENAZI*t, DOUGLAS H. SMITH*, SCOT A. MARSTERS*, LAVON RIDDLES, TIMOTHY J. GREGORYt, DAVID D. Ho§, AND DANIEL J. CAPON¶ Departments of *Immunobiology and tRecovery Process Research, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080; §Aaron Diamond AIDS Research Center, New York University School of Medicine, 455 First Avenue, New York, NY 10016; and $Cell Genesys Inc., 344 Lakeside Drive, Foster City, CA 94404

Communicated by David Botstein, May 17, 1991

ABSTRACT The infection of human cells by laboratory strains of human immunodeficiency virus type 1 (HIV-1) can be blocked readily in vitro by recombinant soluble CD4 and CD4-immunoglobulin hybrid molecules. In contrast, infection by primary isolates of HIV-1 is much less sensitive to blocking in vitro by soluble CD4-based molecules. To investigate the molecular basis for this difference between HIV-1 strains, we isolated the gpl20-encoding genes from several CD4-resistant and CD4-sensitive HIV-1 strains and characterized the CD4bind_ properties of their recombinant gpl20 (rgpl2O) products. Extensive amino acid sequence variation was found between the gpl20 genes of CD4-resistant and CD4-sensitive HIV-1 isolates. However, the CD4-binding affinities of rgpl20 from strains with markedly different CD4 sensitivities were essentially the same, and only small differences were observed in the kinetics of CD4 binding. These results suggest that the lower sensitivity of primary HIV-1 isolates to neutralization by CD4-based molecules is not due to lower binding affinity between soluble CD4 and free gpl20.

(CHO) cells, purified these proteins by immunoaffinity chromatography, and characterized their CD4-binding properties. We show that the CD4-binding affinity of recombinant gp120 (rgpl20) from primary and laboratory HIV-1 strains is essentially the same, suggesting that the difference in CD4 sensitivity of these viruses is independent of their CD4-binding affinity. Small, yet significant, differences were observed in the kinetics of CD4 binding of CD4-resistant vs. CD4sensitive HIV-1 strains, which may contribute to the differential sensitivity to CD4.

METHODS HIV-1 Isolates. Four primary HIV-1 isolates were investigated, three of which (AC-Pl, JM, and JR-CSF) had been shown previously to be dramatically less sensitive to neutralization by soluble CD4 than the laboratory HIV-1 strains IlIb and IIIRF (12). Primary HIV-1 isolates were obtained after a single short-term passage of clinical AIDS patient isolates in normal peripheral blood mononuclear cells (PBMCs). Isolate AC-P1 is from a patient with Kaposi sarcoma, isolates WM and JM are from asymptomatic seropositive individuals, and isolate JR-CSF is an infectious molecular clone derived from a patient with AIDS encephalopathy (13). An additional laboratory isolate, AC-H9, was derived from isolate AC after over a year of cultivation in vitro in H9 cells (12). Neutralization of HIV-1 by CD4-IgG. Neutralization assays were performed as described previously (12). Briefly, 50 tissue culture median infectious dose (TCID50) units of each HIV-1 isolate was incubated with various concentrations of the CD4 immunoadhesin CD4-IgG (see refs. 9 and 11) for 30 min at 37°C and added to 2 x 106 PBMCs from healthy donors and incubated for 7 days, at which time p24 antigen levels were measured. Isolation of gpl20-Encoding Sequences. Sequences encoding gpl20 from AC-Pi, JM, and WM were isolated from PBMCs infected with passage 1 viruses. Uninfected human PBMCs (5 x 106) were cultured with 100 ,ul of infected patient serum, or in the case of AC-H9, with culture supernatant from infected H9 cells. These cultures were tested for p24 antigen and the cells were harvested after the first positive result. Genomic DNA was prepared from the infected cells as described (14), and the gp120-encoding sequences were obtained by PCR (15). Amplifications were done in two steps, with two nested sets of primers based on highly conserved regions of gpl20, as determined by analysis of 20 HIV-1 genomes reported previously (16). The primers were designed to amplify the gp120-encoding sequence of any of

Binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120, to its cellular receptor, CD4, initiates the infection of human mononuclear cells by HIV-1 (1, 2). CD4 is a cell surface glycoprotein found mainly on T lymphocytes, whose normal function is association with class II major histocompatibility molecules on antigen-presenting cells, facilitating antigen recognition by the T-cell receptor. Several recombinant soluble proteins based on the extracellular portion of CD4 have been developed as candidates for AIDS therapy, including soluble CD4 and CD4 immunoadhesins (CD4-immunoglobulin hybrids); these molecules block efficiently the infection of human cells by laboratory strains of HIV-1 in vitro (3-11). Recent studies on the neutralization in vitro of HIV-1 by soluble CD4 have shown that neutralization of primary HIV-1 isolates, in contrast to laboratory strains, requires much higher concentrations of soluble CD4 (12). Primary HIV-1 isolates, nonetheless, can be blocked fully by soluble CD4, as well as by anti-CD4 antibodies. This observation indicates that despite their lower sensitivity to soluble CD4, primary HIV-1 isolates infect cells via a CD4-dependent mechanism, as is the case for laboratory HIV-1 strains. Therefore, it was suggested that the relatively low sensitivity of primary vs. laboratory HIV-1 isolates to the blocking effect of soluble CD4 may be due to a lower CD4-binding affinity (12). To test this hypothesis, we have isolated the gp120encoding sequences from CD4-resistant and CD4-sensitive HIV-1 strains, expressed their recombinant polypeptide products as secreted molecules in Chinese hamster ovary

Abbreviations: HIV-1, human immunodeficiency virus type 1; rgpl2O, recombinant gpl20; PBMCs, peripheral blood mononuclear cells. tTo whom reprint requests should be addressed.

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. 7056

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these HIV-1 strains and thus are unlikely to introduce a significant bias in the isolation of novel gp120 clones. Each primer contained 20 to 22 bases at its 3' end complementary to the gp120 sequences; in the last 18 of these bases there is no more than a single mismatch with any of the 20 sequences, and there is a perfect match for at least 18 of them. The primers also contained restriction sites to allow cloning of the resulting fragments in an expression vector; the 3'-end primers also contained in-frame stop codons. The primer sequences were as follows GGGAATTCGGATCCGAGCAGAAGACAGTGGCAATGA (5' external); GGTCTAGAAGCTTCTACTACATAGTGCYTCCTGCTGCTCC (3' external); GGGAAT-

TCGGATCCGGTACCTGT(G/A)TGGAA(G/A)GAAGCA (5' internal); GGTCTAGAAGCTTCTACTATCC(T/C)AAGAACCCAAGGAACA (3' internal). The gpl20 sequence of JR-CSF was obtained by using a molecular clone of the provirus (13) by PCR amplification with the internal primer pair. Expression of rgpl20. The cloned gp120-encoding sequences were expressed in CHO cells, as described previously for rgpl20 of strain IIIb (17, 18). Cell lines expressing elevated levels of rgpl2W were obtained by dihydrofolate reductase selection and gene amplification in methotrexate. Each resulting rgpl20 polypeptide contains a 25-amino acid leader sequence from the gD glycoprotein of HSV-1 encoded by the vector, which was used to purify the proteins by immunoaffinity chromatography with a monoclonal anti-gD antibody (17, 18). Analysis of CD4-rgpl2O Binding. Equilibrium binding of rgp120 to CD4-IgG was analyzed as described previously (19). Ninety-six-well plates were coated with goat anti-human IgG antibody, blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), incubated with CD4-IgG at 1 tug/ml (1 hr), and washed. In the competition binding assays, these steps were followed by 125I-labeled rgpl20 (1251-rgpl20; 2.5 nM) from strain IIb alone or together with unlabeled rgpl20 at various concentrations (3 hr at 22°C). Nonspecific binding was determined by omitting CD4IgG. Kinetic assays were carried out with 1251-rgpl20 from the IIb or JR-CSF isolates and transfected human 293 cells expressing full-length human CD4 (3). 125I-rgp120 (3 nM) was incubated with 5 x 104 cells in 0.1 ml of PBS/1% BSA containing 10 mM NaN3, for 0-30 min at 37°C (association kinetics). The cells were washed with ice-cold PBS and radioactivity was measured in the cell pellets. For dissociation kinetics assays, the washed cells were resuspended in PBS/1% BSA/10 mM NaN3 containing 0.3 ,uM unlabeled rgpl20 and incubated for 0-120 min at 37°C. The cells were washed and pelleted and radioactivity was measured. Nonspecific binding was determined in the presence of 0.3 ,uM unlabeled rgpl20 added simultaneously with labeled rgpl20. The kinetic data were analyzed according to the following equation (20):

Proc. Natl. Acad. Sci. USA 88 (1991)

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chain constant domains) of human IgG1; the resulting polypeptide folds as a homodimeric protein (9, 11, 21). CD4-IgG has been demonstrated to block infection in vitro by IIb (9, 11). We examined the neutralization of all six HIV-1 isolates by CD4-IgG and found a pattern of neutralization similar to that reported for soluble CD4 (Fig. 1 and ref. 12). The CD4-IgG 50% inhibitory concentrations (IC50 values) were 0.002 ,ug/ml with IIb, 0.023 ,g/ml with AC-H9, 0.7 ,g/ml with AC-P1, 0.6 ,ug/ml with WM, 0.39 ,ug/ml with JM, and 1.6 ,ug/ml with JR-CSF. Thus, the sensitivity of the primary HIV-1 isolates to CD4-IgG was 350- to 800-fold lower than that of IIb, whereas the sensitivity of the laboratory isolate AC-H9 was only 10-fold lower. Notably, the concentrations of CD4-IgG required to block the primary HIV-1 isolates completely were approximately 5-fold lower on a molar basis than those reported for soluble CD4 (10-25 ,ug/ml vs. 50-500 ,ug/ml, with molecular weights of 112,000 and 41,000, respectively) (12). This greater efficiency of CD4-IgG in blocking primary HIV-1 isolates may be due to greater avidity of CD4-IgG resulting from the dimeric structure ofthis molecule as opposed to the monomeric structure of soluble CD4. Amino Acid Sequence of gpl20 from Primary and Laboratory HIV-1 Isolates. High molecular weight DNA was prepared from PBMCs infected with each of the viral patient isolates, and the sequences encoding gpl20 were amplified by PCR. To ensure that the gpl20 clones selected for biochemical analysis were representative of each isolate, 6-12 independent clones were obtained from each patient isolate and characterized by C-track DNA sequence analysis (not shown). For each patient isolate, the two clones showing the greatest number of differences from one another in C-track patterns were selected for complete DNA sequence analysis (Table 1). A low amino acid sequence variation (1-3%) was found between the clones derived from any individual patient (Table 1). This observation confirms the existence of populations of closely related HIV-1 genomes (termed quasispecies) within each patient isolate, which differ slightly in the gpl20-encoding sequence of their env gene, as reported previously for the HIV-1 tat gene (22). A much greater degree of sequence variation (14-23%) was observed between clones from different patients or between the primary and laboratory isolates (Fig. 2 and Table 1). The distribution of sequence differences between the isolates conforms to the pattern of hypervariable and conserved regions reported previously for gpl20 (23). Notably, the divergence between CD4-resistant 100 80-

6 60-

0

ln[Beq/(Beq - Bt)] = (kon[rgp120] + koff)t, where Beq and Bt are bound rgpl20 at equilibrium and at time t, respectively, [rgpl20] is the molar concentration of rgpl20, and kon and koff are the association and dissociation rate

constants, respectively. koff was determined from the linear portion of the dissociation plots (see Fig. 4). In both equilibrium and kinetic assays, nonspecific binding was normally less than 10% of the total binding. Assays were done in triplicate.

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RESULTS AND DISCUSSION Neutralization of Primary and Laboratory Isolates of HIV-1 by CD4-IgG in Vitro. CD4-IgG is composed of the V1 and V2 extracellular domains of human CD4 linked at their carboxyl terminus to the hinge and Fc portion (CH2 and CH3 heavy

FIG. 1. Neutralization of primary and laboratory isolates of HIV-1 by CD4-IgG. Laboratory strains IIb (o) and AC-H9 (o) and primary isolates AC-P1 (e), WM (A), JM (v), and JR-CSF (A) were tested for neutralization by CD4-IgG, using 50 tissue culture median infectious dose (TCIDm) units of virus as the inoculum and 2 x 106 PBMCs as targets.

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may have arisen from a minor component of the virus population in -the original AC isolate. CD4-rpl20 Binding Affinity of Primay and Laboratory EIV-1 Isdiates. The gpl20 clones depicted in Fig. 2 were placed into mammalian expression vectors and stably expressed as secreted proteins in CHO cells, as described previously for rgpl20 from the IIb strain (17, 18). Several micrograms of each rgpl20 was purified to greater than 90% homogeneity by immunoaffinity chromatography and the affinity for CD4-IgG was determined by competition binding analysis (Fig. 3). The Kd for 1'1I-rgp120 from IIb was found to be 3.6 ± 0.2 nM by independent saturation binding analysis (not shown). The competition IC5o value for unlabeled gpl20 from IIlb was 6.3 nM, and the Kd derived from this value was 3.7 nM (Fig. 3), consistent with the results of saturation analysis, and with values reported previously for lIb and cell-surface CD4, soluble CD4, or CD4-IgG (3, 9, 17,21). The ICso for rgpl20 from the other HIV-1 isolates ranged from 4.8 to 7.3 nM, yielding Kd values of 3.1 to 4.7 nM (Fig. 3). Thus, the CD4-binding affinities of rgpl20 from primary and laboratory HIV-1 isolates are essentially the same. This finding suggests that the difference in CD4 sensitivity between primary and laboratory strains is not due to differences in CD4-gpl-20 binding affinity. Long-term propagation of the cloned JR-CSF isolate in PBMCs did not produce CD4-sensitive viruses (data not shown), suggesting that the retrieval of CD4-resistant virus does not depend upon the presence of multiple HIV-1 quasispecies in the virus stocks. While it is possible theoretically that the gp120 sequences cloned from primary HIV-1 isolates

Table 1. Percent identity of deduced amino acid sequences of gp120 cloned from primary and laboratory HIV-1 isolates 8 9 10 1 2 3 4 5 6 7 1 (PV22) 2 (AC-H9.8) 82 3 (AC-H9.10) 83 97 4 (AC-P1.6) 82 82 83 5 (AC-P1.11) 81 82 83 97 85 82 83 83 82 6 (WM.4) 7 (WM.8) 85 81 83 83 82 99 8 (JM.1) 77 78 78 78 77 77 77 9 (JM.2) 77 78 78 78 77 77 77 100 10 (JR-CSF) 86 84 84 81 81 85 85 79 77 Two independent clones from the AC-P1, AC-H9, WM, and JM isolates were sequenced and their comparison is also shown. The sequences were aligned as in Fig. 2.

and CD4-sensitive strains is no greater than that within either group. Indeed, the two strains most different in CD4 sensitivity, IIb and JR-CSF, are as similar in gp120 sequence as any other two strains. Thus, the overall gp12O sequence homology by itself does not appear predictive of sensitivity to soluble CD4. The AC-H9 and AC-P1 isolates, both of which originate in the same patient isolate, are as different from each other as they are from other isolates. This suggests that 1 year of propagation in vitro of AC-H9 may have resulted in a large degree of sequence diversity similar to that observed between isolates from differdnt individuals. Alternatively, AC-H9 PV22 AC-H9. 10 AC-PI .6 tlM.4

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FIG. 2. Protein sequence comparison of gp120 from primary and laboratory isolates of HIV-1. The sequences are aligned with the sequence of the PV22 clone of HIV-1 IIb (16). Only amino acids that differ from those in PV22 are shown for the other isolates. The sequences shown begin at residue 12 of the mature gpl20. The hypervariable domains V1-V5 are indicated.

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Proc. Natl. Acad. Sci. USA 88 (1991)

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gpl20, nM FIG. 3. CD4 binding of recombinant gpl20 from primary and laboratory HIV-1 isolates. Depicted is the residual binding of 125Irgpl2O from IlIb to CD4-IgG at equilibrium in the presence of increasing concentrations of unlabeled rgpl20 from each HIV-1 isolate. n, IIb clone PV22; o, AC-H9 clone 10; *, AC-P1 clone 6; A, WM clone 4; v, JM clone 1; *, JR-CSF. IC50 and Kd values in the competition assay were, respectively (in nM): 6.3, 3.7 (IlIb); 5.0, 3.1 (AC-H9); 7.3, 4.3 (AC-Pl); 7.0, 4.1 (WM); 8.0, 4.7 (JM); and 7.0, 4.1 (JR-CSF). Kd values were based on the equation Kd = IC50/(1 + [TI/[KdTI, where [T] is the concentration of the tracer (2.5 nM) and Kcrr is the Kd of the tracer, as determined by saturation binding (3.6

nM).

might be derived from minor CD4-sensitive virus subpopulations in these patient isolates, this appears unlikely, since multiple gp120 clones from each isolate were characterized and found to be virtually identical by sequence analysis. In addition, there is no reason to believe that our PCR strategy was biased toward selective amplification of CD4-sensitive virus sequences. Moreover, in the case of JR-CSF, in which the same molecular clone was used to test CD4 sensitivity and CD4 affinity, again the binding was found to be the same as for CD4-sensitive isolates. Therefore, we conclude that the differences in CD4 sensitivity between primary and laboratory HIV-1 strains are independent of the binding affinity between soluble CD4 and free gpl20. This conclusion is further supported by recent data showing similar soluble CD4-binding affinities for gpl20 derived by detergent solubilization from intact virions of primary and laboratory HIV-1 strains (John P. Moore and D.P.H., unpublished data). Kinetics of CD4-rgpl2O Binding of Primary and Laboratory HIV-1 Isolates. Since the Kd values were determined at equilibrium, the possibility remained that kinetic differences may exist in the CD4 binding of different isolates, which might not be reflected in the equilibrium constant. To assess

7059

this possibility, we investigated the kinetics of CD4-rgpl2O binding of HIV-1 IIb and JR-CSF, the isolates most differing in CD4 sensitivity (800-fold). Whereas no significant difference in association was observed at 22TC, at 37TC, IIb exhibited significantly faster kinetics of association and dissociation than those exhibited by JR-CSF (Fig. 4). The ko0 and koff rate constants for IIb were, respectively, 2.3 mind1nM-1 and 6.7 min', and for JR-CSF, 1.0 mind nM-' and 2.8 min-. Thus, both the on and off rates were 2.3-fold greater for IIb. The Kd values derived from kff/ko, ratios are 2.9 nM (IIb) and 2.8 nM (JR-CSF), consistent with the values determined by equilibrium binding analysis (Fig. 3). Conclusion. Our results show that despite extensive variation in the gpl20 amino acid sequences of primary and laboratory strains of HIV-1, the CD4-binding affinities of rgpl20 from these viruses are indistinguishable. Thus, assuming that the interaction of rgpl20 with CD4-IgG in vitro reflects accurately the interaction of HIV-1 with soluble CD4 in vivo, the differential CD4 sensitivity of primary and laboratory HIV-1 strains does not appear to be due to differences in CD4-gp120 binding affinity. Small differences were observed between CD4-sensitive and CD4-resistant isolates in the kinetics of CD4-rgpl2O binding. These kinetic differences of about 2-fold in the on and off rate constants appear very small relative to the 800-fold difference in CD4 sensitivity, and therefore they are unlikely to be responsible for the latter difference. Nonetheless, such kinetic differences might be a contributing factor, which, in combination with differences in other events in the process of HIV-1 infection and its blocking by soluble CD4, could lead to differential CD4 sensitivity. A much larger sample of HIV-1 isolates should be investigated, however, and more information should be obtained on the kinetics of cell penetration by HIV-1, to assess further the possibility of a causative relationship between binding kinetics and CD4 sensitivity. The observation that primary HIV-1 isolates are less sensitive than laboratory strains to neutralization by soluble CD4 suggests that distinct selective pressures exist upon HIV-1 in vivo and in vitro that give rise to these different phenotypes. It remains to be seen whether a given CD4 sensitivity phenotype is directly advantageous for growth of HIV-1 in vivo and in vitro or is merely a corollary of another phenotype that provides some growth advantage. Most probably, the genetic determinants that account for the variation in sensitivity to soluble versions of the HIV-1 receptor reside within the env gene, since the products of this gene, gpl20 and gp4l, are crucial to cell attachment and penetration. The finding that primary and laboratory strains of HIV-1 are equally susceptible to blocking by anti-CD4 antibodies (12), taken together with the lack of major differences in CD4-gpl20 binding reported here, suggests that events 1.2-o---

1.0

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FIG. 4. Kinetics of CD4-rgpl2O binding. The association (Left) and dissociation (Right) of'251-rgpl2O from the IlIb (o) orJR-CSF (A) isolates to CD4 were analyzed as described in the text. Beq is the gpl20 binding at equilibrium and Bt is the binding at a given time point.

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which are closely linked to CD4 binding by HIV-1 may contribute to the differential CD4 sensitivity observed. One possible such event is the shedding of gp120 from HIV-1 virions, which can be enhanced by soluble CD4 binding and therefore may contribute to the neutralization of HIV-1 by this molecule (24-27). Alternatively, the interaction of fusogenic sequences in gp41 with the target cell, which appears to follow the interaction ofgp120 with cellular CD4 (reviewed in ref. 28), or the interaction of gp41 with gp120, or the density of gp120 on the virus may differ in primary and laboratory strains. HIV-infected cells can be killed specifically in vitro by cytotoxins or cytotoxic T cells, targeted to the gp120 expressed on the surface of infected cells by soluble CD4-toxin conjugates (29, 30) or hybrid CD4-lgG-anti-CD3 antibodies (31). The finding that rgpl20 from primary HIV-1 strains retains a high CD4 binding affinity suggests that therapeutic strategies based on CD4-targeted killing of virus-infected cells may not be compromised by the apparent resistance of primary HIV-1 strains to soluble CD4. We thank Drs. Rebecca Ward and Chris Clark for helpful discussions, Dr. Irvin Chen for the JR-CSF provirus clone, Dr. Eric Daar for technical assistance, Tom Camerato and Nancy Simpson for help in DNA sequencing, Steve Frie and Dr. Catherine Lucas for help with gpl20 radioimmunoassays, and Carol Morita for graphics. 1. Sattentau, Q. J. & Weiss, R. A. (1988) Cell 52, 631-633. 2. Robey, E. & Axel, R. (1990) Cell 60, 697-700. 3. Smith, D. H., Byrn, R. A., Marsters, S. A., Gregory, T., Groopman, J. E. & Capon, D. J. (1987) Science 328, 17041707. 4. Fisher, R. A., Bertonis, J. M., Meier, W., Johnson, V. A., Schooley, D. S. & Flavell, R. A. (1988) Nature (London) 331,

76-78.

5. Hussey, R. E., Richardson, N. E., Kowalski, M., Brown, N. R., Chang, H., Siliciano, R. F., Dorfman, T., Walker, B., Sodroski, J. & Reinherz, E. L. (1988) Nature (London) 331, 79-81. 6. Deen, K. C., McDougal, S., Inacker, R., Folena-Wasserman, G., Arthos, J., Rosenberg, J., Maddon, P. J., Axel, R. & Sweet, R. W. (1988) Nature (London) 33, 82-84. 7. Traunecker, A., Luke, W. & Karjalainen, K. (1988) Nature (London) 33, 84-86. 8. Nara, P. L., Hwang, K. M., Rausch, D. M., Lifson, J. D. & Eiden, L. E. (1989) Proc. Natl. Acad. Sci. USA 86, 7139-7143. 9. Capon, D. J., Chamow, S. M., Mordenti, J., Marsters, S. A., Gregory, T., Mitsuya, H., Byrn, R. A., Lucas, C., Wurm, F. M., Groopman, J. E., Broder, S. & Smith, D. H. (1989) Nature (London) 337, 525-531. 10. Traunecker, A., Schneider, J., Kiefer, H. & Karjalainen, K. (1989) Nature (London) 339, 68-70. 11. Byrn, R. A., Mordenti, J., Lucas, C., Smith, D. H., Marsters,

Proc. Natl. Acad. Sci. USA 88 (1991)

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