The serum concentrations of CH01-like and VRC-CH31-like antibodies were 70.4 8.1 (mean standard deviation) CH01 g/ml equivalents and 42 10.5 VRC-CH31 g/ml equivalents, respectively. 3 neutralizers) (46,19,20). It is unclear if such plasma neutralization breadth is mediated by many (17) or only one or a few (23,24) antibody specificities. A number of bnAbs have been isolated from HIV-1-infected individuals, and these include those directed to a variable STA-21 loop 1 and 2 (V1V2) conformational (quaternary) epitope, to the CD4-binding site (CD4bs), and to outer domain glycans, all on the gp120 surface unit of the HIV-1 envelope glycoprotein (2,3,21,22,24). Additional bnAbs target the membrane-proximal external region (MPER) of gp41 (13,26). While evidence suggests that bnAbs to the gp41 MPER are limited by tolerance mechanisms, the V1V2 conformational and CD4bs antibodies are generally less polyreactive (2,8,10,12,22). However, they both present unusual characteristics: the V1V2 conformational bnAbs have long heavy-chain complementarity-determining region 3 (HCDR3) (2,10,14,15), whereas the CD4bs bnAbs display a high degree of somatic mutation (10,12,17,18,24) and appear to derive from restricted VHgene families (18,24). Strategies that allow for highly specific serologic and/or neutralization assays to determine the epitopes of plasma bnAbs are now well established (1,6,9,23), and recent studies suggest that a single HIV-1-infected subject can STA-21 make bnAbs of multiple specificities (20). If both V1V2 conformational and CD4bs antibodies could be isolated from the same individual and demonstrated to recapitulate the serum neutralizing activity, this would provide direct evidence in support of a polyvalent bnAb HIV-1 vaccine strategy. For this study, we selected a chronically STA-21 infected individual (CH0219) whose plasma displays extraordinary broad and potent neutralization and includes antibody specificities directed against MPER, CD4bs, CD4-induced (CD4i), gp120 core, and V1V2 conformational epitopes (20). While responses against the CD4bs and a PG9-like V1V2 conformational epitope appeared to be responsible for most of the breadth, only a limited number of mapping reagents were available to confirm this observation (20). Because polyvalent neutralizing antibody responses may be a key consideration for HIV-1 vaccine development, and considering the exceptional breath of serum neutralization in this subject, we interrogated the IgG+memory B-cell repertoire of donor CH0219 to isolate and characterize the antibodies that recapitulated serum neutralization. TSHR By using a clonal memory B-cell culture system (2), we previously identified four bnAbs (CH01, CH02, CH03, and CH04), members of the same clonal lineage, binding to a V1V2 conformational epitope (2). The CH01 through CH04 bnAbs neutralized 36% to 46% of 91 cross-clade HIV-1 isolates, which represented a subset of strains also neutralized by PG9 (2). Another clone of five CD4bs-specific bnAbs (VRC-CH30, VRC-CH31, VRC-CH32, VRC-CH33, and VRC-CH34) was isolated from the same donor by antigen-specific B-cell sorting of individual IgG+memory B cells reactive with RSC3 but not RSC3371 (25). VRC-CH30 through VRC-CH34 neutralized 75% to 95% of a multisubtype panel of viruses with breadth comparable to that of VRC01 (25).Figure 1shows the phylogenetic tree of the VRC-CH30 to -CH34 bnAb clonal lineage. Analysis of the V(D)J rearrangements of the CH01 to CH04 and VRC-CH30 to -CH34 sequences demonstrated that the two clones use distinct VHgenes (VH3 and VH1, respectively) (Table 1), that the frequency of somatic mutations of the VRC-CH30 to -CH34 VHchains (23 to 24%) is approximately twice that of CH01 to CH04 (12 to 14%) (Table 1), and that, conversely, CH01 to CH04 have substantially longer HCDR3s (24 amino acids [aa] versus 13 aa, according to the Kabat numbering system [7]) (Table 1). These data demonstrate that the two clones did not share a genetic background, relative to VHgenes, and suggest that they likely evolved independently. == Fig 1. == Phylogenetic tree of the VRC-CH30 to VRC-CH34 monoclonal antibodies. The tree shows the evolutionary distances of the V(D)J nucleotide sequences of the VRC-CH30 to VRC-CH34 monoclonal antibodies and is rooted on the nucleotide sequence of the unmutated common ancestor (UCA) sequence. It is important to note the great distance of each of the mature antibodies from the common UCA sequence, as we previously observed for the CH01.