Vol. 65, No. 3

JOURNAL OF VIROLOGY, Mar. 1991, p. 1370-1376 0022-538X/91/031370-07$02.00/0 Copyright © 1991, American Society for Microbiology

Common Immunologic Determinant between Human Immunodeficiency Virus Type 1 gp4l and Astrocytes MASAHITO YAMADA,' ANDREAS ZURBRIGGEN,l MICHAEL B. A. OLDSTONE, 2 AND ROBERT S. FUJINAMIlt* Department of Pathology, University of California, San Diego, La Jolla, California 92093,1 and Division of Virology, Department of Neuropharmacology, Research Institute of Scripps Clinic, La Jolla, California 920372 Received 16 August 1990/Accepted 14 November 1990

Monoclonal antibodies against a synthetic 12-amino-acid peptide that comprises the immunodominant domain of human immunodeficiency virus type 1 gp4l (amino acids 598 through 609) reacted with astrocytes found in human and rodent central nervous system tissue. The monoclonal antibodies bound to a 43-kDa protein found in central nervous system tissue preparations. These results indicate that human immunodeficiency virus type 1 gp4l contains a common epitope with astrocytes and that an immune response to human immunodeficiency virus type 1 gp4l could generate antibodies that are cross-reactive to astrocytes. Furthermore, anti-astrocyte antibodies, which were directed at a common epitope with the gp4l sequence, were found to be present in cerebrospinal fluid from some AIDS patients with central nervous system complications. Astrocytes regulate the environment for appropriate neuronal function, and astrocyte hyperactivity (astrocytosis) is known to be the common and early pathologic event in brains from patients with central nervous system AIDS. We suggest that antibody-induced effect(s) on astrocytes could lead to the physiologic neuronal dysfunctions observed in AIDS patients.

Here, we report that MAbs against a synthetic 12-aminoacid peptide of HIV gp4l (amino acids 598 through 609) reacted with astrocytes in human and rodent CNS tissue. The MAbs bound to a 43-kDa CNS protein from noninfected tissue. Further, cerebrospinal fluid (CSF) from some AIDS patients with CNS complications contained anti-astrocyte antibodies that were directed at a common epitope with the gp4l sequence. These results indicate the presence of a common determinant between HIV-1 gp4l and astrocytes. We suggest that the generation of such a cross-reactive antibody or antibodies to astrocytes by HIV infection could contribute to the CNS complications found in AIDS patients.

AIDS dementia complex (ADC) is a common syndrome with neuropsychiatric complications of infection with human immunodeficiency virus type 1 (HIV-1) (6, 29-31). Recent studies of brains from ADC patients have demonstrated HIV-1 infection of the brain only in a subset of patients; HIV-1 was rarely observed in neuronal cells, suggesting that the pathogenesis of ADC is unlikely to be simply and solely attributable to direct HIV-1 infection (2, 31, 39). Further, the reversibility of ADC with 3'-azido-3'-deoxythymidine therapy (40) indicates that the lesion is not likely structural but more likely neuropharmacologic or metabolic. We have proposed that viruses can initiate autoimmune disease through molecular mimicry via common or crossreacting determinants (8). Previously, we found that monoclonal antibodies (MAbs) to viral proteins from both DNA and RNA viruses could bind to host cell components (4, 10, 35). Subsequently, the biologic importance of molecular mimicry was established by demonstrating the ability to initiate an autoimmune disease with a viral peptide that had sequence similarity with a disease-inducing region (7). To determine whether immune responses to HIV-1 could cross-react with central nervous system (CNS) tissue and contribute to CNS complications such as ADC through a mechanism of HIV-1-induced autoimmunity, we focused on the amino acid sequence 598 through 609 (Leu-Gly-Ile-TrpGly-Cys-Ser-Gly-Lys-Leu-Ile-Cys), the transmembrane portion of HIV-1 gp4l. This region was investigated because (i) almost all patients with neurological complications of AIDS had antibodies to HIV-1 gp4l protein, not to other viral proteins, and intra-blood-brain barrier (BBB) production of the antibodies was observed (33); and (ii) an immunodominant region for HIV-1 was mapped to gp4l, and sera from HIV-1-infected patients reacted with this sequence (11-13).

MATERIALS AND METHODS Production of MAbs. BALB/c mice, which were maintained in the Research Institute of Scripps Clinic, were immunized with a HIV-1 gp4l peptide (amino acids 598 through 609) coupled to keyhole limpet hemocyanin in adjuvant. Several immunizations were performed until the immunized mice demonstrated antibody to gp4l in sera. Thereafter, these mice were boosted with peptide conjugate on days -3, -2, and -1 before removal of spleens for hybridoma production (19). Supernatant fluid from hybridoma-containing wells was screened by an enzyme-linked immunosorbent assay (34) with the gp4l peptide. Lines producing antibody to the peptide and having reactivity to HIV by enzyme-linked immunosorbent assay were identified and cloned three times by limiting dilution. Thereafter, MAbs were obtained from culture supernatants or ascitic fluids. Immunohistochemistry with the MAbs. CNS tissues from normal mice or rats and from mice with pathological conditions including murine relapsing experimental allergic encephalomyelitis (1), Theiler murine encephalomyelitis virus infection (23), mouse brain with a wound injury, and human cerebral infarction were examined. All mice and rats were

* Corresponding author. t Present address: Department of Neurology (3R210), University

of Utah Medical Center, 50 N. Medical Dr., Salt Lake City, UT 84132. 1370

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COMMON DETERMINANT BETWEEN HIV-1 AND ASTROCYTES

perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Human brain from a 36-year-old man with cerebral infarction was obtained at autopsy 1 h after his death and fixed in buffered 10% Formalin. All tissues were embedded in paraffin. Staining was performed by the avidinbiotin-peroxidase complex (ABC) method (17). Sections were treated sequentially with the following: (i) proteinase K (Bethesda Research Laboratories, Gaithersburg, Md.), 5 ,ug/ml in 50 mM Tris (pH 7.5)-2 mM CaCl2 for 15 min at room temperature (RT); (ii) 5% normal goat serum in PBS for 30 min at RT; (iii) the MAbs for 2 h at 37°C; (iv) 1:40 biotinylated horse anti-mouse immunoglobulin G (IgG; Vector, Burlingame, Calif.) for 30 min at RT; (v) ABC (Vector) for 30 min at RT; and (vi) diaminobenzidine (Sigma, St. Louis, Mo.)-H202. In the experiments designed to block positive reactions, the MAb was preincubated with 1 mM gp4l peptide or an irrelevant peptide in PBS overnight at 4°C before immunostaining. Double labeling with the MAb and antibody to GFAP. To

identify the brain cells that were positive for the MAbs, double labeling was performed (27). Since gp4l epitopepositive cells were morphologically similar to astrocytes, an antibody to glial fibrillary acidic protein (GFAP) was used as a cell marker (3). (i) Double immunofluorescent staining. Sections were incubated sequentially with (i) a mixture of the MAb (1:500) and rabbit antibody to GFAP (1:100; Dako, Santa Barbara, Calif.) for 2 h at 37°C and (ii) a mixture of fluorescein isothiocyanate-conjugated goat antibody (IgG fraction) to mouse IgG (1:40; Cooper Biomedical, Malvern, Pa.) and rhodamine-conjugated goat antibody (IgG fraction) to rabbit IgG (1:40; Cooper) for 30 min at 37°C. Results were observed with a fluorescent microscope. (ii) Double immunoperoxidase staining. Sections were first stained with the MAb by the ABC method (17) and then with antibody to GFAP by the peroxidase-antiperoxidase method (17). Briefly, after ABC staining with the MAb as described above, sections were incubated sequentially with (i) antibody to GFAP (1:300) for 2 h at 37°C, (ii) goat anti-rabbit IgG (1:50; Dako) for 30 min at RT, (iii) peroxidase-antiperoxidase complex (1:50; Dako) for 30 min at RT, and (iv) 0.05% 4-chloro-1-naphthol (Sigma) and 0.01% H202 in Tris-saline (pH 7.6) for 20 min at RT. The reciprocal experiment was also performed to confirm results. Western immunoblot analysis. Samples were obtained from mouse brains 3 weeks after needle injury and from gliotic brain tissue surgically removed from a patient with epilepsy. The samples were homogenized in 0.15 M Tris (pH 6.7)-3% sodium dodecyl sulfate-30% glycerol at a concentration of 0.2 g/ml and centrifuged for 2 min at 5,400 x g. The supernatant fluid was adjusted to 0.005% bromophenol blue and 1% 2-mercaptoethanol, and samples were boiled for 2 min. Aliquots of the brain homogenate containing 200 ,ug of total protein were electrophoresed on 10.5% polyacrylamide gels with 2.5% polyacrylamide stacking gels at 150 V (10). Separated proteins in the polyacrylamide gel were electrophoretically transferred to nitrocellulose paper at 150 mA for 18 h (10). Immunoreactions were performed as follows: (i) blocking with 3% bovine serum albumin in PBS for 30 min, (ii) incubating with the MAbs (1:100 dilution) for 3 h at RT, (iii) washing with 1.5% bovine serum albumin in PBS, (iv) incubating in 1251I-labeled sheep anti-mouse immunoglobulin (Amersham, Arlington Heights, Ill.) in 3% bovine serum albumin with 106 cpm per tube for 1 h at RT, (v) washing and drying, and (vi) autoradiographing (10). In inhibition experiments, the MAb was preincubated with gp4l peptide over-

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TABLE 1. MAbs to HIV-1 gp4l peptide MAb

IgG subclass

Titer (log2)a

M-1 M-2 M-4 M-6 M-11 M-13 M-22 M-24 M-25 M-28 M-29 M-36

1 or 2b 2b 2b 2b 1 2b 2b 1 1 1 1 1

17.4 17.6 12.9 16.8 22.5 6.8 5.3 5.3 6.3 5.3 19.0 20.0

Tissue reactivityb

+ + + +++ ++ + ++

-

a Titer of ascitic fluid to gp4l peptide by enzyme-linked immunosorbent assay. b Reactivities with CNS astrocytes by immunohistochemistry. Degrees: -, absent; +, mild; + +, moderate; + + +, marked. The MAb with the strongest reactivity, M-22, stained brain cells at high dilutions, i.e., 1:100,000, when ascitic fluid was used.

night at 4°C before the immunoreactions were performed. As negative controls, strips were incubated with only a labeled second antibody or an irrelevant first antibody followed by a second antibody. Immunohistochemistry of CSF samples from the patients. CSF samples were obtained from 14 patients with ADC, 3 AIDS patients with myelopathy, 1 AIDS patient with seizure, 11 HIV-positive patients without neurologic involvement, and 10 HIV-negative controls. Staining was performed by the ABC method described above. Sections were incubated with undiluted CSF samples for 16 h at 4°C. Then sections were incubated sequentially with 1:40 biotinylated anti-human IgG (Vector) and with ABC and reacted with diaminobenzidine-H202. The results of immunohistochemical studies in each sample were evaluated without knowledge of the clinical information. In the experiments designed to block positive reactions, the CSF sample was preincubated with 1 mM gp4l peptide or an irrelevant peptide (myelin basic protein peptide) in PBS overnight at 4°C before

immunostaining. RESULTS AND DISCUSSION

MAbs were raised against a synthetic HIV gp4l peptide (amino acids 598 through 609), and the reactivity of the MAbs to CNS tissues was examined by immunohistochemistry. The MAbs used in this study are shown in Table 1. In immunohistochemical studies, cells reacting with the MAbs to gp4l were observed in normal CNS tissues of mice (Fig. la) or rats (Fig. lb) and in diseased CNS tissues including murine relapsing experimental allergic encephalomyelitis (Fig. lc), encephalomyelitis induced by Theiler murine encephalomyelitis virus, mouse brain with a wound injury, and human cerebral infarction (Fig. le). In normal CNS tissue, gp4l epitope-positive cells were scattered in subpial and subependymal portions (Fig. la and b). The cytoplasm, including cellular processes, was stained, but the rounded or oval nuclei were spared. The gp4l epitopepositive cells increased in number in CNS tissues obtained from pathologic lesions as described above (Fig. lc and e). The cells in the lesions had thick cellular processes (Fig. lc) and a hypertrophic perikaryon (Fig. le) and were morphologically consistent with reactive astrocytes. Preincubation of the MAbs with the gp4l peptide, but not with an irrelevant

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Common immunologic determinant between human immunodeficiency virus type 1 gp41 and astrocytes.

Monoclonal antibodies against a synthetic 12-amino-acid peptide that comprises the immunodominant domain of human immunodeficiency virus type 1 gp41 (...
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