Antibodies to CD4 in Individuals Infected with Human Immunodeficiency Virus Type 1

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 3346-3350, May 1989 Medical Sciences Antibodies to CD4 in individuals infected with human immunodeficiency virus type 1 (acquired immunodeficiency syndrome/autoantibodies/virus receptor/glycoprotein gpl20-binding region) MARK KOWALSKI*, BLAIR ARDMANt, LADAN BASIRIPOUR*, YICHEN Lu*, DIETMAR BLOHM*, WILLIAM HASELTINE*t, AND JOSEPH SODROSKI*§ *Division of Human Retrovirology, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, and tDepartment of Cancer Biology, Harvard School of Public Health, Boston, MA 02115; and tDepartment of Medicine, New England Medical Center, Boston, MA 02111 Communicated by Matthew D. Scharff, December 23, 1988 (receivedfor review November 11, 1988)

ABSTRACT The attachment of human immunodeficiency against the extracellular region of CD4. These antibodies virus type 1 (HIV-1) to target cells is mediated by a specific were shown to recognize a CD4 region distinct from that interaction between the viral envelope glycoprotein (gpl20) and important for gpl20 binding and, thus, are unlikely to arise by the CD4 receptor. Here we report that approximately 10% of an anti-idiotypic response to gpl20. The potential role of HIV-1-infected individuals produce antibodies that recognize anti-receptor antibodies in the pathogenesis of AIDS is the extracellular portion of the CD4 molecule. Carboxyl- discussed. terminal deletions of CD4 that do not affect HIV-1 gpl20 binding eliminate recognition of CD4 by patient antisera. In MATERIALS AND METHODS contrast, mutations in the amino-terminal domain of CD4 that attenuate HIV-1 gpl20 binding do not diminish CD4 recogni- Plasmids. The plasmid pCD4, which expresses a soluble tion by patient antisera. These results suggest that HIV-1 form of CD4, was constructed by cloning the BamHI frag- infection can generate antibodies directed against a region of ment of the plasmid pAc373/T4ex described previously (12) the viral receptor distinct from the virus-binding domain. into an expression vector that utilizes the cytomegalovirus early promoter and contains simian virus 40 origin of repli- cation. This BamHI fragment includes a stop codon imme- Human immunodeficiency virus type 1 (HIV-1) is the etio- diately 5' to the sequences encoding the transmembrane logic agent of acquired immunodeficiency syndrome (AIDS) region. Soluble HIV-1 gpl20 was expressed by using the (1, 2). Depletion of CD4+ lymphocytes can account for most plasmid pIIIenvA517 described previously (40). ofthe severe immunologic abnormalities in AIDS patients (3) Construction of CD4 Mutants. Linker insertion mutagene- and occurs prior to, or coincident with, the development of sis of CD4 was performed by partial digestion of pCD4 with severe opportunistic infections in most HIV-i-infected indi- restriction enzymes (Alu I, Hinfl, Sty I, Pvu II, Stu I, Bcl I), viduals (4-6). creation ofblunt ends by using the Klenow fragment ofDNA The tropism of HIV-1 is determined by a specific interac- polymerase I if necessary, and ligation of appropriately sized tion between the gpl20 exterior-envelope glycoprotein and synthetic linkers (New England Biolabs) to preserve the CD4 the CD4 molecule, an integral membrane protein that serves reading frame or to introduce a desired frameshift (in the as a receptor for the virus (7-10). The CD4 protein consists 182S, 148S, and 118S mutants). The position of the linker of an amino-terminal extracellular portion (approximately insert was analyzed by restriction enzyme digestion and 372 amino acids long), a transmembrane region, and an DNA sequence analysis. intracytoplasmic region. Soluble CD4 molecules containing Binding of Soluble CD4 to HIlV-1 gp120. COS-1 cells were only the extracellular portion ofCD4 retain the ability to bind transfected with 5-10 ,ug of the CD4-expressing plasmid and to the HIV-1 gpl20 glycoprotein (11-16). CD4 regions im- 5,ug ofpIIIenvA517 as described (41). Forty-eight hours after portant for gpl20 binding have been localized to amino acids transfection, the cells were metabolically labeled overnight 40-56 within the amino-terminal immunoglobulin-like do- with 100 ,Ci of [35S]cysteine and 30 ,Ci of [3H]leucine main (17-21). (New England Nuclear; 1 ,Ci = 37 kBq) per ml in 3.0 ml of HIV-1 infection of CD4+ lymphocytes in vitro is accom- cysteine- and leucine-free Dulbecco's modified Eagle's me- panied by virus-mediated cytopathic effects (1, 2, 22-24). dium (GIBCO). The culture supernatants containing radio- Immune-mediated mechanisms involving antibody-depen- labeled soluble CD4 and soluble gpl20 were collected, cell dent cytotoxicity (25-28) or cytotoxic lymphocytes (29) have debris was removed by low-speed centrifugation, and the also been demonstrated to be capable of CD4+ lymphocyte supernatants were precleared with 100 Al of protein A- destruction in vitro. The contribution ofthese processes to in Sepharose beads (Pharmacia) without coupled antibody. vivo CD4+ lymphocyte depletion in AIDS is unknown. Other Coimmunoprecipitation and sodium dodecyl sulfate/poly- immunologic mechanisms for CD4+ lymphocyte destruction acrylamide gel electrophoresis (SDS/PAGE) of the bound have been suggested. Anti-lymphocyte antibodies, some of complexes of CD4 and gpl20 were performed with either which bind to CD4+ lymphocytes, have been detected in the OKT4 (Ortho) or serum from an HIV-1-seropositive individ- sera of HIV-infected individuals (30-34). It has been pro- ual (19501) as described (40). posed that anti-gp120 antibodies produced in response to Radioimmunoprecipitation of Soluble CD4 by Patient Sera. HIV-1 infection might elicit anti-idiotypic antibodies that Aliquots ofCOS-1 cell supernatants containing metabolically mimic gp120 and bind to CD4 (35-39). Using a soluble form labeled, soluble CD4 or gpl20 were treated with either 3 IlI ofCD4, we demonstrate that approximately 10%o of sera from of human serum or 5 Al of a purified anti-CD4 monoclonal HIV-1-infected individuals contains antibodies directed antibody. The mixtures were incubated for 18 hr at 4°C, followed by the addition of 30 ,ul of protein A-Sepharose The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: HIV-1, human immunodeficiency virus type 1. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 3346 Downloaded by guest on September 28, 2021 Medical Sciences: Kowalski et al. Proc. Natl. Acad. Sci. USA 86 (1989) 3347 beads (Pharmacia). After a further 2-hr incubation, the protein was not precipitated from supernatants of cells immunoprecipitate-bead complexes were washed six times transfected with the pCD4 plasmid by an antibody that does with a detergent buffer containing 0.5% Nonidet P-40 and not recognize CD4. 0.5% sodium deoxycholate. The immunocomplexes were Radiolabeled supernatants from transfected COS-1 cells eluted from the beads by boiling in a SDS-containing buffer were used for immunoprecipitation by human sera obtained and analyzed by PAGE under nonreducing conditions. The from HIV-1-infected individuals and from control subjects. immunoprecipitated proteins were visualized by autoradiog- The HIV-infected individuals included male homosexuals, raphy of the enhanced and dried gels. intravenous drug abusers, and hemophiliacs and represented Immunoglobulin Purification. Human sera were absorbed clinical states from healthy to active AIDS. The control to protein A-Sepharose beads to purify the IgG-containing subjects were either healthy individuals or individuals hos- fraction. The purified IgG was then size-fractionated by fast pitalized for a variety of non-HIV-related illnesses, including protein liquid chromatography on a Superose 12 column autoimmune disorders. Ofthe 91 sera from HIV-seropositive (Pharmacia) to separate aggregated immunoglobulins and subjects tested, 9 (10%) immunoprecipitated a 46-kDa protein immunocomplexes (>170 kDa) that may have been present from supernatants of pCD4-transfected, but not mock- from noncomplexed IgG (<170 kDa). The noncomplexed transfected, COS-1 cells (Fig. 1 and Table 1). The sera that IgG-containing fractions were concentrated by vacuum dial- demonstrated reactivity to the 46-kDa protein were derived ysis and then used for immunoprecipitation as described from seropositive homosexual males as well as intravenous above. drug abusers. Statistically significant correlations with dis- ease status were not observed in this study. None ofthe sera from 112 HIV-seronegative individuals immunoprecipitated RESULTS a 46-kDa protein from pCD4-transfected COS-1 cell super- Antibodies Reactive to Soluble CD4. To examine human natants (Table 1). sera for the presence of anti-CD4 antibodies, a soluble To investigate whether the 46-kDa protein precipitated by molecule consisting of the entire extracellular portion of the the sera of some HIV-1-infected individuals corresponded to native CD4 glycoprotein was used. The recombinant CD4 the soluble CD4 glycoprotein, the radiolabeled cell superna- glycoprotein was produced by transfection of COS-1 cells tants from pCD4-transfected COS-1 cells were first immu- with the pCD4 plasmid. This plasmid contains a CD4 cDNA noprecipitated with either OKT4A or an irrelevant monoclo- with a stop codon immediately 5' to the sequences encoding nal antibody, UPC-10. The OKT4A- and UPC-10-precleared the transmembrane region (42). After transfection of COS-1 supernatants were then used for immunoprecipitation by cells with the pCD4 plasmid, precipitation ofradiolabeled cell serum from a HIV-1-infected subject that had demonstrated supernatants with OKT4, OKT4A, or anti-Leu-3A monoclo- reactivity to the 46-kDa protein. Fig. 1B shows that preclear- nal antibodies, which recognize different CD4 epitopes, ing the pCD4-transfected COS-1 cell supernatants with the revealed a 46-kDa protein on non-reducing gels not present in OKT4A but not with the UPC-10 monoclonal antibody mock-transfected cell supernatants (Fig. LA). The 46-kDa removed the 46-kDa protein recognized by the serum from a HIV-infected individual. These results suggest that the au- A thentic soluble CD4 glycoprotein is recognized by the serum 1 2 3 4 5 6 7 8 9 10 1 1 12 13 of some HIV-infected individuals. Because it was possible that the anti-CD4 reactivity in the *.- patient sera was due to gpl2O-anti-gpl2O immunocomplexes 46- _ Table 1. Immunoprecipitation of soluble CD4 by human antisera HIV No. positive/ serological Clinical AIDS risk no. testedt B status* status group (% positive) 1 2 3 4 5 6 7 8 9 10 11 12 13 Seronegative Healthy Homosexual 0/20 (0%o) Healthy NA 0/25 (0%) 46 - ..em Malignant NA 0/15 (0%o) disease Autoimmune NA 0/37 (0O) disease Unknown NA 0/15 (0O) FIG. 1. Immunoprecipitation of soluble CD4 by sera from HIV- infected individuals. (A) Radiolabeled cell supernatants from mock- 0/112 (0%o) transfected (lanes 1 and 13) or pCD4-transfected (lanes 2-12) COS-1 cells were immunoprecipitated with OKT4 (lanes 1 and 2), anti- Seropositive Healthy Homosexual 2/15 (13%) Leu-3A (lanes 12 and 13), or sera from HIV-positive individuals ARC Homosexual 3/19 (16%) (lanes 3-11). The sera immunoprecipitating the soluble CD4 product AIDS Homosexual 1/12 (8%) were 57 (lane 5), 526-1 (lane 8), and RV119 (lane 11). (B) Radiolabeled Healthy Hemophiliac 0/5 cell supernatants from mock-transfected (lanes 1 and 2) COS-1 cells (0%) were immunoprecipitated with OKT4A monoclonal antibody (lane 1) AIDS Hemophiliac 0/1 (0%o) or RV119 patient serum (lane 2). Labeled cell supernatants from Healthy i.v. drug user 0/1 (0%) pCD4-transfected COS-1 cells (lanes 3-13) were repeatedly pre- ARC i.v. drug user 2/24 (8%) cleared by immunoprecipitation with either OKT4A (lanes 7-11 AIDS i.v. drug user 1/14 (7%) represent successive immunoprecipitations) or UPC-10, a control (lo0) monoclonal antibody (lanes 12 and 13 represent the first and fifth 9/91 immunoprecipitations). The supernatants that had been precleared ARC, AIDS-related complex; NA, not applicable. with OKT4A (lanes 3 and 4) or UPC-10 (lanes 5 and 6) were then used *HIV serological status was determined by Western blot or radio- for immunoprecipitation by serum from a healthy seronegative immunoprecipitation analysis. subject (lanes 3 and 5) or from a HIV-seropositive patient, RV119 tSera were tested for reactivity to soluble CD4 by immunoprecip- (lanes 4 and 6). The radioimmunoprecipitates were run on SDS/ itation oflabeled pCD4-transfected COS-1 cell supernatants using 3 polyacrylamide gels under nonreducing conditions to prevent dis- pl ofpatient sera as described. Numbers in parentheses refer to the tortion of the soluble CD4 band observed with some human sera. percentage of positive tests. Downloaded by guest on September 28, 2021 3348 Medical Sciences: Kowalski et al. Proc. Natl. Acad. Sci. USA 86 (1989)

A 7 8 9 1 0 1 1 1 2 1 31 41 5 16 1 71 8 size fraction that was coeluted with purified human IgG 1 2 3 4 5 6 (s170 kDa) was able to radioimmunoprecipitate the soluble CD4 product from supernatants of pCD4-transfected COS-1 cells (data not shown). These results suggest that the reac- 50-i ...h liv tivity to soluble CD4 of some HIV-positive sera resides in the 50- _ immunoglobulin G fraction. CD4 Regions Important for gpl20 Binding. A possible mechanism for the generation of anti-CD4 autoantibodies in HIV-infected individuals is an anti-idiotypic response elicited B by antibodies directed against the CD4-binding region of the 1 2 3 4 9 10 1 112 HIV gpl20 glycoprotein (35-39). This model predicts that the anti-CD4 autoantibodies mimic the gpl20 glycoprotein and, 12 0 1 5 6 7 8 120 therefore, should bind to the CD4 regions utilized by gp120 for binding. To test this prediction, mutants in the soluble CD4 protein were compared for ability to bind gpl20 and for recognition by patient sera. Ne -50 To test CD4 mutants for binding to the HIV gpl20 50- glycoprotein, a transient expression system was used to produce soluble gp120 and CD4 glycoproteins. The plasmid pIIIenvA517 (40) was used to express a soluble HIV-1 gpl20 FIG. 2. HIV-1 gp120 binding ability of soluble CD4 linker- molecule in the supernatants of COS-1 cells cotransfected insertion mutants. (A) Radioimmunoprecipitates of supernatants with the mutant CD4 plasmid. The radiolabeled supernatants from COS-1 cells that were mock-transfected (lanes 1 and 12) or transfected with pCD4 alone (lanes 2 and 13), pIIIenvA517 alone were precipitated with OKT4, OKT4A, or a HIV-i-positive (lanes 7 and 14), pCD4 and pIIIenvA517 (lanes 3, 8, and 15), pCD4-18 serum (19501) that does not contain anti-CD4 antibodies. and pIIIenvA517 (lanes 4, 9, and 16), pCD4-178 and pIIIenvA517 Binding of mutant soluble CD4 proteins to HIV-1 gp120 was (lanes 5, 10, and 17), or pCD4-212 and pIIIenvA517 (lanes 6, 11, and assessed by the reciprocal coprecipitation of the two mole- 18). Immunoprecipitations were performed with OKT4 (lanes 1-6), cules by using OKT4 or 19501 antibodies. Specificity of the OKT4A (lanes 7-11), or 19501 patient serum (lanes 12-18). (B) coprecipitation was assured by lack of precipitation of gpl20 Radioimmunoprecipitates of supernatants from COS-1 cells that by the OKT4A antibody, which competes with gpl20 for CD4 were transfected with pIIIenvA517 only (lanes 1, 5, and 9), pCD4 and (Fig. 2A) (9). pIIIenvA517 (lanes 2, 6, and 10), pCD4-50 and pIIIenvA517 (lanes 3, Most of the CD4 mutants created retained the ability to 7, and 11), or pCD4-56 and pIIIenvA517 (lanes 4, 8, and 12). 2A Table Two of the linker Immunoprecipitations were performed with OKT4 (lanes 1-4), bind to gpl20 (Fig. and 2). OKT4A (lanes 5-8), or 19501 patient serum (lanes 9-12). insertion mutations specifically resulted in a decreased as- sociation of the soluble CD4 protein with the HIV-1 gpl20 rather than true anti-CD4 antibodies, the IgG fraction of a (Fig. 2B and Table 2). These mutations are located in the first patient serum was isolated and tested for anti-CD4 reactivity. extracellular CD4 domain (residues 50 and 56). The pheno- For this purpose, immunoglobulin from one positive patient types of these CD4 mutants are consistent with the obser- serum was purified by binding to protein A-Sepharose, vations of others that CD4 sequences in this region are followed by size fractionation on a Superose-12 column. A important for gp120 binding (17-21). Table 2. gpl20 binding and HIV-1-positive sera recognition of soluble CD4 mutants gpl20 OKT4A Patient sera Plasmid Predicted amino acid sequence binding recognition recognition Linker insertion mutants pCD4 Wild-type + + + pCD4-18 (16)CTASQ -* CTAHRWASQ + + + pCD4-50 (49)SKLN -- SKPIDGLN - + + pCD4-56 (54)NDRAD -- NDRPSMGAD - + + pCD4-63 (62)WDQG -* WDQASMQG + - + pCD4-72 (71)IKNL -* IKNPIDGNL + + ND pCD4-131 (130)CRSP - CRSPIDGSP + + ND pCD4-150 (149)LELQ LEPIDGLQ + + + pCD4-178 (176)VLAFQ -- VLAHRWAFQ + + + pCD4-182 (181)KASS KAHRWASS + + + pCD4-206 (205)EKLT EKPIDGLT + + + pCD4-212 (211)GELW - GEPIDGLW + + l pCD4-296 (295)TQLQ - TQPIDGLQ + + 4 Truncated mutants 182S (80)QKAS. .. QKAIDGLQHSL* + + - 148S (147)SQLE. .. SQPSMAGAPG* + 4 _

118S (116)LTLE. . . LTLPSRCKG* + 4 - The radiolabeled COS-1 cell supernatants containing soluble gp120 and mutant CD4 proteins were immunoprecipitated with an AIDS patient serum (19501), OKT4, or OKT4A. The level of expression of conformationally intact CD4 mutant protein was assessed by the OKT4 and OKT4A immunoprecip- itation. The amount of CD4 mutant protein coprecipitating with gpl20 was judged relative to the level of expression of that mutant protein to obtain an estimate of gpl20 binding. The values reported represent the results of a minimum of three separate experiments: +, 100% of that observed with wild type; 4, decreased relative to wild type; -, negative; ND, not determined. The numbers in parentheses shown in the predicted amino acid sequence (shown in single-letter code) refer to the residue that is amino-terminal. Asterisks denote a stop codon. Downloaded by guest on September 28, 2021 Medical Sciences: Kowalski et al. Proc. Natl. Acad. Sci. USA 86 (1989) 3349 Stop codons were introduced into the CD4 coding se- approximately 10% of HIV-1-infected individuals. The pres- quences of the pCD4 plasmid to produce soluble CD4 ence ofanti-CD4 antibodies appeared to be specific for HIV-1 proteins with carboxyl-terminal truncations. The truncated infection. None of the 112 seronegative control subjects soluble CD4 products were tested for the ability to bind to the exhibited anti-CD4 antibodies in their sera, even though a HIV-1 gpl20 glycoprotein. For this purpose, radiolabeled substantial fraction of this group had either autoimmune supernatants of COS-1 cells cotransfected with plasmid diseases or belonged to a high risk group for HIV infection. expressing a truncated CD4 protein and pIIIenvAl517 were The anti-CD4 reactivity observed is not likely due to the precipitated with 19501 patient serum. Truncated CD4 prod- presence of antibody-gpl20 complexes in the patient sera. ucts containing 182, 148, or 118 residues of the CD4 protein This assertion follows from the observation that purified were precipitated by the 19501 serum only in the presence of immunoglobulin G is capable ofprecipitating the soluble CD4 soluble HIV-1 gpl20 (Table 2). We interpret these data to protein and that the CD4 regions important for the serum mean that the 182S, 148S, and 118S truncated products still reactivity do not correspond to those regions important for bind to gpl20 and that the anti-gp120 antibodies in the 19501 gpl20 binding. serum (which does not recognize CD4) precipitates the The patient serum reactivity is dependent upon sequences gpl20-truncated CD4 complexes. Therefore, sequences suf- in the carboxyl-terminal half of the extracellular portion of ficient for gp120 binding are located in the amino-terminal 118 residues of the human CD4 glycoprotein. CD4. These CD4 sequences are dispensible for binding to the Patient Antibody Recognition of Soluble CD4 Mutants. To HIV gpl20 envelope glycoprotein. These results indicate that define CD4 regions important for recognition by some HIV- the anti-CD4 antibodies do not represent anti-idiotypes 1-positive sera, soluble CD4 molecules containing linker- elicited by anti-gp120 antibodies. Anti-idiotypic antibodies insertion or deletion mutations were produced by COS-1 cell that mimic gpl20 should bind to the gpl2O-binding domain of transfection. Radiolabeled supernatants were precipitated CD4. The mechanism for generating the anti-CD4 antibodies with patient sera that were known to react with the parental is unknown, but might involve the exposure of novel CD4 soluble CD4 product. epitopes as a result of gpl20-CD4 interaction or as a result Linker-insertion mutations in the amino-terminal half of of CD4' lymphocyte destruction in the course of HIV the soluble CD4 molecule did not affect the ability of the infection. patient sera to immunoprecipitate the protein (Fig. 3). In Do anti-CD4 antibodies influence the pathogenesis of particular, linker-insertion mutations that attenuated gpl20- quantitative or qualitative T-lymphocyte abnormalities in binding activity or OKT4A recognition did not disrupt HIV-1-infected individuals? The lack of detectable anti-CD4 sequences important for reactivity of five HIV-1-positive antibodies in the majority of HIV-infected subjects suggests sera tested (Figs. 2 and 3 and data not shown). These results that such antibodies are not required for CD4+ lymphocyte suggest that at least some of the sequences important for depletion. However, it is possible that some HIV-sero- anti-CD4 antibody recognition are distinct from those impor- positive individuals harbor anti-CD4 autoantibodies that are tant for gpl20 binding. bound to CD4+ lymphocytes andt thus, are undetectable in While OKT4A recognizes the truncated CD4 protein that our assays. Although an immunopathologic function of anti- contains 182 amino-terminal residues (see the 20-kDa band in CD4 antibodies cannot be addressed without functional lane 3 of Fig. 3A), a HIV-1-positive serum that immunopre- studies, anti-CD4 antibodies do not appear to be sufficient for cipitated the parental soluble CD4 did not precipitate the the induction of clinically significant immunologic abnormal- truncated CD4 protein (Fig. 3B, lane 3). A total of five ities, since some of the individuals with these antibodies are HIV-positive sera that reacted with the parental soluble CD4 healthy. Thus, our findings suggest that other processes are protein were tested. None demonstrated the ability to pre- responsible for CD4+ lymphocyte depletion in the majority of cipitate the 182-amino acid product (data not shown). These HIV-1-infected individuals. results suggest that CD4 sequences carboxyl-terminal to These results raise the possibility that virus infections in amino acid 182, which are not necessary for gpI20 binding, general may elicit changes in immune-system recognition of are important host cell molecules intimately associated with the viral for recognition by anti-CD4 antibodies. exterior proteins. While a role in the pathogenesis of AIDS remains to be demonstrated, anti-receptor antibodies gener- DISCUSSION ated during the course of infection with other viruses may lead to clinically significant sequelae. In the studies reported herein, serum antibodies directed The presence of anti-CD4 autoantibodies might influence against the extracellular portion of CD4 were detected in the pharmacokinetics of soluble CD4 analogues administered as a treatment for AIDS. The half-life and perhaps therapeu- B tic efficacy may be altered for CD4 analogues that retain

3 6 7 8 1 234 5 6 7 8 epitopes recognized by anti-CD4 antibodies. 46- We thank Bruce Walker, Robert Schooley, David Ho, Eleanor Levy, Paul Demchak, and Mary Fran McLane for gifts of sera; Ellis Reinherz for the gift of the pAc373/T4ex plasmid; and John Bristol for help with some of the experiments. This work was supported by the National Institutes of Health (AI24755, A127729 and FOD0757), 20- the American Foundation for AIDS Research, and the Leukemia Society of America. B.A. was supported by a fellowship from the John A. Hartford Foundation, Inc. FIG. 3. Immunoprecipitation of mutant CD4 proteins by a patient serum reactive with soluble CD4. Immunoprecipitation of trans- 1. Broder, S. & Gallo, R. (1984) N. Engl. J. Med. 311, 1292-1297. fected COS-1 cell supernatants with either OKT4A antibody (A) or 2. Barre-Sinoussi, F., Chermann, J., Rey, F., Nugeyre, M. T., a positive patient serum (serum 63) (B). The supernatants were Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Brun- derived from COS-1 cells that were mock-transfected (lane 1) or Vezinet, F., Rouzioux, C., Rozenbaum, W. & Montagnier, L. transfected with pCD4 (lane 2), pCD4-182S (lane 3), pCD4-50 (lane (1983) Science 220, 868-870. 4), pCD4-56 (lane 5), pCD4-18 (lane 6), pCD4-212 (lane 7), or 3. Fauci, A. S., Macher, A. M., Longo, D. L., Lane, H. C., pCD4-296 (lane 8). Analysis of the immunoprecipitates was per- Rook, A. H., Masur, H. & Gelman, E. P. (1984)Ann. let. Med. formed on SDS/polyacrylamide gels using nonreducing conditions. 100, 92-106. Downloaded by guest on September 28, 2021 3350 Medical Sciences: Kowalski et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4. Gottlieb, M. S., Schroff, R., Schanker, H. M., Weisman, Natl. Acad. Sci. USA 83, 4297-4301. J. D., Fan, P. T., Wolf, R. A. & Saxon, A. (1981) N. Engl. J. 23. Sodroski, J., Goh, W. C., Rosen, C., Campbell, K. & Hasel- Med. 305, 1425-1431. tine, W. A. (1986) Nature (London) 322, 470-474. 5. Lane, H. C., Masur, H., Gelman, E. P., Longo, D. L., Steis, 24. Lifson, J. D., Feinberg, M. B., Reyes, G. R., Rabin, L., R. G., Chused, T., Whalen, G., Edgar, L. C. & Fauci, A. S. Banapour, B., Chakrabarti, S., Moss, B., Wong-Staal, F., (1985) Am. J. Med. 78, 417-422. Steimer, K. S. & Engleman, E. G. (1986) Nature (London) 323, 6. Friedman-Kien, A. E., Laubenstein, L. F., Rubenstein, P., 725-727. Buimovici-Klein, E., Marmur, M., Stahl, R., Spigland, I., Kim, 25. Lyerly, H. K., Matthews, T. J., Langlois, A. J., Bolognesi, D. P. & Weinhold, K. J. (1987) Proc. Natl. Acad. Sci. USA 84, K. S. & Zolla-Pazner, S. (1982) Ann. Int. Med. 96, 693-699. 4601-4605. 7. Dalgleish, A. G., Beverly, P. C., Clapham, P. R., Crawford, 26. Blumberg, R. S., Paradis, T., Hartshorn, K. L., Vogt, M., Ho, D. H., Greaves, M. G. & Weiss, R. A. (1984) Nature (London) D. D., Hirsch, M. S., Leban, J., Sato, V. L. & Schooley, R. T. 312, 763-766. (1987) J. Infect. Dis. 156, 878-884. 8. Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., 27. Ljunggren, K., Bottiger, B., Biberfeld, G., Karlson, A., Fenyo, Guetard, D., Hercend, T., Gluckman, J. & Montagnier, L. E. M. & Jondal, M. (1987) J. Immunol. 139, 2263-2267. (1984) Nature (London) 312, 767-771. 28. Ojo-Amaize, E. A., Nishanian, P., Keith, D. E., Houghton, 9. McDougal, J. S., Kennedy, M. S., Sligh, J. M., Cort, S. P., R. L., Heitjan, D. F., Fahey, J. L. & Giorgi, J. V. (1987) J. Mawle, A. & Nicholson, J. K. A. (1986) Science 231, 382-385. Immunol. 139, 2458-2463. 10. Maddon, P. J., Dalgleish, A. G., McDougal, J. S., Clapham, 29. Siliciano, R. F., Lawton, T., Knall, C., Karr, R. W., Berman, P. R., Weiss, R. A. & Axel, R. (1986) Cell 47, 333-348. P., Gregory, T. & Reinherz, E. L. (1988) Cell 54, 561-575. 11. Fisher, R. A., Bertonis, J. M., Meier, W., Johnson, V., Costo- 30. Kloster, B. E., Tomar, R. H. & Spira, T. J. (1984) Clin. poulos, D. S., Lin, T., Tizard, R., Walker, B. D., Hirsch, Immunol. Immunopathol. 33, 330-335. M. S., Schooley, R. T. & Flavell, R. (1988) Nature (London) 31. Dorsett, B., Cronin, W., Chuma, V. & loachim, H. L. (1985) 331, 76-78. Am. J. Med. 78, 621-626. 12. Hussey, R. E., Richardson, N. E., Kowalski, M., Brown, 32. Ozturk, G. E., Kohler, P. F., Horsburgh, C. R. & Kirkpatrick, N. R., Change, H., Siliciano, R. F., Dorfman, T., Walker, B., C. H. (1987) J. Clin. Immunol. 7, 130-139. Sodroski, J. & Reinherz, E. (1988) Nature (London) 331, 78-81. 33. Stricker, R. B., McHugh, T. M., Moody, D. J., Morrow, 13. Berger, E. A., Fuerst, T. R. & Moss, B. (1988) Proc. Nati. W. J. W., Stites, D. P., Shuman, M. A. & Levy, J. A. (1987) Acad. Sci. USA 85, 2357-2361. Nature (London) 327, 710-713. 14. Smith, D., Byrn, R., Marsters, S., Gregory, T.,- Groopman, 34. Warren, R. Q., Johnson, E. A., Donnelly, R. P., Lavia, M. F. J. E. & Capon, D. J. (1987) Science 238, 1704-1707. & Tsang, K. Y. (1988) Clin. Exp. Immunol. 73, 168-173. 15. Deen, K., McDougal, S. J., Inacker, R., Folena-Wasserman, 35. Ziegler, J. L. & Stites, D. P. (1986) Immunol. Immunopathol. G., Arthos, J., Rosenberg, J., Maddon, P. J., Axel, R. & 41, 305-313. Sweet, R. W. (1988) Nature (London) 331, 82-84. 36. Martinez, A. C., DeLattera, A., Alonso, J. M., Marcos, 16. Traunecker, A., Luke, W. & Karjalainen, K. (1988) Nature M. A. R., Marquez, C., Toribio, M. L. & Coutinho, M. L. (London) 331, 84-86. (1988) Lancet i, 454-457. 17. Jameson, B., Rao, P., Kong, L., Hahn, B. H., Shaw, G. M., 37. McDougal, J. S., Nicholson, J. K. A., Cross, D. G., Cort, Hood, L. W. & Kent, S. B. (1988) Science 240, 1335-1339. S. P., Kennedy, M. S. & Mawle, A. C. (1986) J. Immunol. 137, 18. Clayton, L. K., Hussey, R. E., Steinbrich, R., Ramachandran, 2937-2944. H., Husain, Y. & Reinherz, E. L. (1988) Nature (London) 335, 38. Chanh, T. C., Dreesman, G. R. & Kennedy, R. C. (1987) Proc. 363-366. Natl. Acad. Sci. USA 84, 3891-3895. 19. Landau, N. R., Warton, M. & Littman, D. R. (1988) Nature 39. Dalgleish, A. G., Chanh, T. C., Thomson, B. J., Malkovsky, (London) 334, 159-162. M. & Kennedy, R. C. (1987) Lancet Hi, 1047-1049. 20. Richardson, N. E., Brown, N. R., Hussey, R. E., Vaid, A., 40. Kowalski, M., Potz, J., Basiripour, L., Dorfman, T., Goh, Matthews, T. H., Bolognesi, D. P. & Reinherz, E. L. (1988) W. C., Terwilliger, E., Dayton, A., Rosen, C., Haseltine, W. Proc. Nati. Acad. Sci. USA 85, 6102-6103. & Sodroski, J. (1987) Science 237, 1351-1355. 21. Peterson, A. & Seed, B. (1988) Cell 54, 65-72. 41. Cullen, B. R. (1987) Methods Enzymol. 152, 684-704. 22. DeRossi, A., Franchini, G., Aldovini, A., Del Mistro, A., 42. Maddon, P. J., Littman, D. R., Godfrey, M., Maddon, D. E., Chieco-Bianchi, L., Gallo, R. C. & Wong-Staal, F. (1986) Proc. Chess, L. & Axel, R. (1985) Cell 42, 93-104. Downloaded by guest on September 28, 2021