The structures of 447-52D and 537-10D in complex with V3 peptides show that two of the three structural determinants the long CDR H3 making main-chain interactions with the N-terminus of V3 crown, and the docking of the relatively conserved GPG region of the V3 crown can maximally tolerate the sequence variation that occurs in the central portion of the V3 loop. its epitopes. Designing immunogens that can elicit broadly neutralizing antibodies able to overcome this diversity remains a major challenge in developing an effective HIV vaccine. The virus surface glycoprotein gp120 has five variable regions, named V1-V5, that exhibit high sequence diversities. Among them, V3 is relatively conserved due to its functional importance in the life cycle of the virus it determines the viral co-receptor usage and viral tropism of the virus by binding to the virus co-receptors on the cell surface, CCR5 or CXCR4, a critical step in viral Allopregnanolone entry (Ivanoff et al., 1992;McKeating et al., 1989;Resch et al., 2001;Wyatt and Sodroski, 1998). The V3 conservation is reflected both in its sequence and structure. V3 has limited variation in length; it is almost always 35 amino acids long, and it harbors at its tip a highly conserved motif: Gly-Pro-Gly-Arg/Gln (GPGR/Q, residues 312-315 in the HXB2 numbering scheme,Ratner et al., 1987). While the GPGR containing V3 (V3GPGR) is predominant in clade B viruses, V3GPGQis more often found in clade C and other non-clade B viruses (Kuiken et al., 1999). V3 is structurally characterized in the context of the gp120 core (Huang et al., 2007;Huang et al., 2005). It protrudes ~30 from the CD4-bound gp120 core and this extended structure can be divided into 3 regions: the base, the stem and the crown. The conserved base is seated in the gp120 core with a disulfide bridge (Leonard et al., 1990), while the stem extends outwards and is flexible (Huang et al., 2007;Huang et al., 2005). The crown has a unique beta-conformation with the GPGR/Q turn at the distal apex of the crown. Interestingly, all the known human anti-V3 monoclonal antibodies (mAbs) are against the crown (Gorny et al., 1993;Zolla-Pazner et al., 2004;Zwart et NPM1 al., 1991), and studies have shown that the crown is accessible on the surface of most HIV-1 primary isolates and serves as a neutralization epitope (Gorny et al., 2004). V3 is highly immunogenic and was named the principal neutralizing determinant of HIV-1 (Javaherian et al., 1989). Anti-V3 antibodies are found to exist in Allopregnanolone the sera of essentially all patients infected with HIV-1 (Carrow et al., 1991;Krachmarov et al., 2005;Krachmarov et al., 2001;Vogel et al., 1994), and immunization of animals with V3-containing immunogens can elicit anti-V3 immune responses (Javaherian Allopregnanolone et al., 1990;Law et al., 2007;Zolla-Pazner et al., 2008). Importantly, a novel immunization regimen combining DNA primes and protein boosts has recently been shown to focus the rabbit immune response on the V3 epitope and elicit antibodies with cross-clade neutralization capacities (Zolla-Pazner et al., 2009;Zolla-Pazner et al., 2008). Moreover, there are monoclonal and polyclonal anti-V3 antibodies display both intraand inter-subtype neutralization of diverse HIV strains (Binley et al., 2004;Conley et al., 1994;Gorny et al., 1992;Krachmarov et al., 2005). Thus, the V3 epitope is well-suited as a target for HIV-1 vaccine development (Zolla-Pazner, 2004). Structural characterization of anti-HIV-1 human mAbs targeting immunogenic epitopes on the surface glycoprotein gp120 of HIV-1 can potentially facilitate the design and development of an effective HIV-1 vaccine (Burton, 1997;Douek et al., 2006;Zolla-Pazner, 2004). A structure-aided reverse-engineering strategy of immunogen design requires (i) identification of broadly reactive human mAbs, (ii) determination of their structures.