HIV-1 infection induces neutralizing (Nt) antibodies (Abs) against one's infecting virus, yet rarely do such Abs neutralize a broad viral spectrum. A few broadly(b)-Nt Abs have been discovered: 4E10, 2F5, b12, 447-52D and 2G12; all but 2G12 have longer-than-usual CDR-H3 hypervariable loops (H3s). Typically, H3 is crucial to antigen contact; yet the crystal structures of 4E10, 2F5 and 447-52D show that, while prominent, their H3s either contact marginal residues of their peptide-epitopes or do not contact these epitopes at all. Haynes et al. (2005) reported that most of the bNt MAbs bind to self-antigens (e.g., 2F5 and 4E10 bind cardiolipin; CL), and speculated they are autoAbs that arose through a process of positive selection on autoantigen followed by recruitment by Env into the adaptive Ab response. Under this hypothesis, bNt Abs arise when tolerance is broken, allowing self-reactive B cells to survive, and Abs with long H3s to emerge. Thus, it was proposed that, to produce bNt Abs, HIV-1 vaccines must break tolerance and produce self-reactive Abs. Our preliminary studies indicate that if 4E10 and 2F5 are autoAbs they are unusual, as their affinity for CL is 100-1000-fold weaker than for their HIV-1 peptide epitopes. We also did not detect increased CL reactivity in 12 bNt sera from HIV+ donors nor in 20 other HIV+ sera, compared to HIV- controls. As it is claimed that long H3s reflect a loss of tolerance, we produced a DNA sequence database of 450 human MAbs of known specificity, obtained mainly from people with autoimmune disease, chronic and acute infection, and found that long H3s exist mainly among anti-protein subsets of the autoimmune and chronic Abs; among the latter, long-H3 Abs are not restricted to HIV infection. From this we propose 3 hypotheses: (1) Long-H3 Abs are produced during HIV-1 infection through loss of tolerance during B-cell development. This predicts that long-H3 Abs accumulate in the IgM+/IgD+, mature, naive B cell compartment, and among IgG+ memory B cells; bNt sera should have significant autoreactivity. (2) Long-H3 Abs arise during antigen-driven processes. These Abs should be present only in the IgG+ memory B cell and plasma cell compartments, and bNt sera should not to be autoreactive. (3) HIV antigens select long-H3 Abs from normal Ab repertoires; this has the same predictions as (2). To test these alternatives, we plan to analyze sera and PMBCs collected from 12 untreated HIV+ donors whose sera are bNt, 12 HIV+ donors whose sera are not broadly neutralizing, 12 patients with SLE, and 14 healthy donors. B cells will be sorted by phenotype into naive, memory, and plasma cell pools and if possible into single, HIV-specific B and plasma cells; expressed mu or gamma VH genes from each B cell pool and from single cells will be sequenced and analyzed for H3 length, somatic mutations, and gene usage. Sera and Abs produced by antigen-specific cells will be analyzed on microarrays bearing autoantigens, HIV-1 antigens, and markers of polyreactivity. We expect to determine if long-H3 Abs are present in B-cell subsets in HIV-1 infection vs. a truly autoimmune state, and whether distinct autoreactivities or generalized polyreactivity is present in bNt sera. The results should help to clarify if HIV vaccines should be designed to break tolerance. [unreadable] [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: One approach to AIDS vaccine design uses HIV-1-neutralizing antibodies. These antibodies are rare and their structures are considered unusual; moreover, questions have arisen concerning their ability to react with self . The proposed work will address whether HIV-neutralizing antibodies are unusual and/or self-reactive , and should clarify whether induction of unusual, self-reactive antibodies is a necessary consideration in AIDS vaccine design. [unreadable] [unreadable] [unreadable]