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 CD4- bind_ properties of their recombinant gpl20 (rgpl2O) prod- ucts. 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. 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 extracel- lular portion of CD4 have been developed as candidates for AIDS therapy, including soluble CD4 and CD4 immunoad- hesins (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 gp120- encoding sequences from CD4-resistant and CD4-sensitive HIV-1 strains, expressed their recombinant polypeptide products as secreted molecules in Chinese hamster ovary (CHO) cells, purified these proteins by immunoaffinity chro- matography, 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 essen- tially the same, suggesting that the difference in CD4 sensi- tivity 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. CD4- sensitive HIV-1 strains, which may contribute to the differ- ential sensitivity to CD4. METHODS HIV-1 Isolates. Four primary HIV-1 isolates were investi- gated, three of which (AC-Pl, JM, and JR-CSF) had been shown previously to be dramatically less sensitive to neutral- ization 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 lab- oratory 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 encod- ing 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 ob- tained 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 de- signed to amplify the gp120-encoding sequence of any of Abbreviations: HIV-1, human immunodeficiency virus type 1; rgpl2O, recombinant gpl20; PBMCs, peripheral blood mononuclear cells. tTo whom reprint requests should be addressed. 7056 The publication costs of this article were defrayed in part by page charge payment. 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