Structure solution identified one Fab per asymmetric unit in a P41212 lattice. bind a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural contamination. Binding to this new site of vulnerability may thus be an important complement to current mAb-based approaches to immunotherapies, prophylaxis, and vaccine design. Induction of a potent neutralizing antibody response capable of recognizing highly diverse isolates of HIV-1 is among the very most important goals of HIV vaccine research. This represents a considerable challenge given the extraordinary antigenic variability of the Env surface glycoprotein. However, approximately 20% of the HIV-infected population does develop a humoral immune response capable of recognizing highly diverse strains2C6. In the past several years improved patient cohorts2C6, HIV-specific B cell isolation7C9, and IgG cloning techniques 10,11 have permitted extraordinary progress in isolation of broadly neutralizing monoclonal antibodies (bNabs) from these individuals. Thus far, these primarily fall into four categories based upon the position of their epitopes around the Env protein, a trimer of gp120 and gp41 heterodimers that is the target of neutralizing antibodies. These sites include the CD4-binding site on gp1208,12 (of which VRC01 is an example), the glycan-containing regions of V1V2 on gp120 (of which PG9 and PG16 are examples), the V3 region centered on the N332 glycan of gp1207,13 (of which PGT121 AICAR phosphate is an example) and the membrane-proximal external region (MPER) on gp41 (of which 10E8 is an example)14,15. It remains unclear to what extent these four categories represent the prevalent and immunodominant sites of Env vulnerability through which broad neutralizing responses are mediated, or whether additional specificities exist16C19 Here we report the isolation of a broad and potently neutralizing HIV-specific mAb, 35O22, that binds a novel epitope. The neutralizing AICAR phosphate activity of 35O22 is usually highly complementary to the activities of other known bNabs. We used mutagenesis, crystallography and EM to define the Env site targeted by 35O22. Our results indicate that 35O22 neutralization occurs by a novel mode of trimer recognition along a conserved face on contiguous areas of gp41 and gp120. To further understand the specificities that underlie broadly neutralizing antibody responses we applied a technique to identify human mAbs of interest from peripheral blood B cells without prior knowledge of the target specificity9. IgG+ B cells of a donor (N152), with broad and potent neutralizing serum and from whom recently described 10E8 antibody was cloned20, were sorted and expanded. The supernatants of B cell microcultures were screened for neutralizing activity and IgG genes from positive wells were cloned and re-expressed. In addition to the 10E8 antibody, 8 clonal family variants of an additional antibody with neutralization activity were found, among which the 35O22 antibody was the most potent and broad (Supplementary Table 1a,b). This antibody was derived from and germline genes, and was highly somatically mutated in variable genes of both heavy chain (35%) and light chain (24%) compared to germline. The 35O22 antibody possessed a heavy-chain complementarity-determining 3 region (CDR H3) composed of 14 amino acids (Fig. 1a and Supplementary Table 2) and an insertion of 8 amino acids in framework 3 (FR3). High levels of somatic mutation AICAR phosphate and FR3 insertions are features of other HIV-specific bNabs7C9,12,13,21,22. Autoreactivity or CD58 polyreactivity are properties of several HIV-specific antibodies23, 24 that could limit their use in therapies or prophylaxis. However, 35O22 bound Hep-2 epithelial cells only modestly (Extended Data Fig. 1a) and did not bind a panel of autoantigens (Extended Data Fig. 1b,c). Against a large panel of pseudoviruses, 35O22 neutralized 62% of 181 isolates with an IC50<50 g/ml (Fig..