Cardiolipin Polyspecific Autoreactivity in Two Broadly Neutralizing HIV-1 Antibodies Barton F. Haynes, 1 * Judith Fleming, 1 E. William St. Clair, 1 Herman Katinger, 2 Gabriela Stiegler, 2 Renate Kunert, 2 James Robinson, 3 Richard M. Scearce, 1 Kelly Plonk, 1 Herman F. Staats, 1 Thomas L. Ortel, 1 Hua-Xin Liao, 1 S. Munir Alam 1 The design of a human immunodeficiency virus–1 (HIV-1) immunogen that can induce broadly reactive neutralizing antibodies is a major goal of HIV-1 vaccine development. Although rare human monoclonal antibodies (mAbs) exist that broadly neutralize HIV-1, HIV-1 envelope immunogens do not induce these antibody specificities. Here we demonstrate that the two most broadly reactive HIV-1 envelope gp41 human mAbs, 2F5 and 4E10, are polyspecific autoantibodies reactive with the phospholipid cardiolipin. Thus, current HIV-1 vaccines may not induce these types of antibodies because of autoantigen mimicry of the conserved membrane-proximal epitopes of the virus. These results may have important implications for generating effective neutralizing antibody responses by using HIV-1 vaccines. A major obstacle to generating a successful human immunodeficiency virus–1 (HIV-1) vac- cine is the inability to induce broadly reactive neutralizing antibodies after immunization with HIV-1 envelope proteins (Env) (1). HIV-1 in- fection and vaccination induce multiple types of antibodies, including antibodies to envelope variable loops, the CD4 binding site, and the chemokine receptor binding site. However, these antibodies do not control HIV-1 and are readily escaped by the virus (1). The human monoclonal antibodies (mAbs) 2F5 and 4E10 represent rare antibodies with broadly neutral- izing activity made from B cells of HIV-1– infected humans, which react with conserved membrane-proximal amino acids in HIV-1 gp41 (2–4). Both antibodies have relatively long hydrophobic CDR3 regions (3, 4). A major conundrum has been that HIV-1 enve- lopes that express membrane-proximal epitopes fail to induce equivalent antibodies in animal models or humans (5–8). Long hydrophobic CDR3 regions are generally typical of poly- reactive autoantibodies (9), and HIV-1–infected patient B lymphocytes are driven to make poly- clonal antibodies with reactivity to the endog- enous phospholipid, cardiolipin (10). In light of these observations, we assayed 2F5 and 4E10 mAbs, along with other mAbs to HIV-1, for cardiolipin and other autoantigen reactivities. The mAbs 2F5 and 4E10, two additional rare broadly reactive neutralizing mAbs (2G12 and IgG1b12) (11–13), and 31 common human mAbs to HIV-1 Env were tested for reactivity with cardiolipin (14) (Table 1). Both 2F5 and 4E10 reacted with cardiolipin, whereas all 33 of the other human mAbs did not react. mAb 2F5 also reacted with histones and the centromere B autoantigen, whereas mAb 4E10 reacted with the systemic lupus erythematosus (SLE) auto- Fig. 1. Reactivity of 2F5, 4E10, and IgG1b12 mAbs with human HEp-2 epithelial cells. (A) mAb 2F5 reacting with HEp-2 cells in a diffuse cytoplasmic and nuclear pattern. (B) mAb 4E10 reacting with HEp-2 cells in a pattern similar to 2F5. (C) mAb IgG1b12 re- acting with HEp-2 cells in a diffuse cyto- plasmic pattern, with nucleoli reactive in the nucleus. (Inset) Higher magnification of cells showing the nucleolar reactivity of IgG1b12 (arrows). (D) Negative reactivity of mAb 1.9F on HEp-2 cells. Anti- body amounts per slide assayed in [(A) to (D)] were 3.75 mg per slide (150 mg/ml) of mAb. mAb 2F5 was positive on HEp-2 cells at 0.125 mg per slide (5 mg/ml). mAb 4E10 was positive on HEp-2 at 0.125 mg per slide (5 mg/ml), and IgG1b12 was positive at 1.25 mg per slide (50 mg/ml). All panels are shown at magnification 200; the inset in (C) is 400. Images shown are from an experiment representative of three performed. 1 Duke University School of Medicine, Durham, NC 27710, USA. 2 Institute of Applied Microbiology, University of Agriculture, Vienna, Austria. 3 Tulane University School of Medicine, New Orleans, LA 70112, USA. *To whom correspondence should be addressed. E-mail: hayne002@mc.duke.edu R EPORTS 24 JUNE 2005 VOL 308 SCIENCE www.sciencemag.org 1906