Schematic diagram of trimeric HIV-1 Env with sites of epitopes for broadly neutralizing antibodies. The four general specificities for BnAbs detected to date are: the CD4 binding site, the V1/V2 variable loops, certain exposed glycans and the MPER. Red ovals, gp120 core; dark red ovals, V1/V2 loops; magenta ovals, V3 loop; blue and red squares, gp41; bright red stripe, MPER of gp41; light brown curved stripe, viral membrane bilayer. PGT antibodies⁶⁸ and 2G12 depend on Env N-linked glycans for binding gp120, as do V1/V2-directed conformational antibodies^31,71.

Clonal tree illustrating the inference scheme.

B-cell ontogeny and the locations of obstacles B cells must overcome to make broadly neutralizing antibodies. Human B cells arise from committed pro-B cells that proliferate in response to hematopoietic growth factors and rearrange IGH V, D and J gene segments (Box 2). After assembly of the pre-BCR, pre-B I cell numbers expand through proliferation and exit the cell cycle as pre-B II cells. Increased expression of the V(D)J recombinase in pre-B II cells drives light-chain gene rearrangements and the assembly of mature BCRs that are capable of binding antigen. B cells in this primary repertoire with long BCR HCDR3s are often autoreactive, and many of these and other autoreactive cells are lost in the bone marrow at the first tolerance checkpoint (as in 2F5 BnAb mice^38,39); the remainder of the B cells mature as T1 and T2 B cells, which migrate into the peripheral lymphoid tissues via the blood. In the periphery, T2, or newly formed, B cells are subject to another round of immune tolerization (tolerance checkpoint 2) before entering the mature B-cell pools. At each of the tolerance checkpoints, the number of autoreactive B cells is reduced by half. Mature B cells activated by antigen and T_FH cells form germinal centers (GCs), which are sites of intense B-cell proliferation, AID-dependent somatic hypermutation, class-switch recombination and affinity-driven selection. A portion of the mutated germinal center B cells acquire new autoreactivity as a consequence of this process of mutation and selection, and some of these cells may become anergic (tolerance checkpoint 3). For serum antibody levels to persist, long-lived plasma cells in bone marrow or elsewhere must be induced. Although the precise location of the long-lived plasma cells is under debate, the HIV-1 envelope is probably a poor inducer of these cells because the durations of the envelope responses in HIV-1 vaccination are generally short lived compared to those in other vaccinations^96,97. Recent data suggest that human plasma cells are less autoreactive than memory B cells³⁵. B cells that make BnAbs must survive tolerance checkpoints 1 and 2 and must also be selected for activation and expansion. The affinity of antigen binding to BCRs is one determinant of B-cell survival and expansion in germinal centers^26–29. Most HIV-1 BnAbs are very heavily somatically mutated, indicating a requirement for persistent antigen drive and complicated antigen-maturation pathways that are probably driven by multiple antigens. BnAbs that recognize the gp41 MPER frequently have VH1-69 as the heavy-chain variable domain⁷³, and CD4 binding site BnAbs frequently come from VH1-2 or VH1-46 genes^70,72. This restricted VH usage for CD4 binding site BnAbs may derive from the requirement for selection of VH-VL pairs that, after extensive somatic hypermutation and affinity maturation, can form an antigen-combining site that resembles CD4 (ref. 70). HCDR2, contained within V_H, corresponds to the gp120 contact loop in CD4. Imm. B, immature B cells.

Steps of a B-cell-lineage–based approach to vaccine design. Step 1 is to isolate V_H and V_L chain members from the peripheral blood or tissues of patients containing BnAbs and to express these native Ig chain pairs as whole antibodies. Step 2 is to infer intermediate ancestor antibodies (IAs, labeled 1, 2 and 3) and the unmutated ancestor antibody (UA) (Box 3). Step 3 requires producing the unmutated and intermediate ancestors as recombinant mAbs and using structure-based alterations in the antigen (changes in Env constructs predicted to enhance binding to the unmutated or intermediate ancestors) or deriving altered antigens using a suitably designed selection strategy. Vaccine administration might prime with the antigen that binds the unmutated ancestor most tightly, and this is then followed by sequential boosts with antigens optimized for binding to each intermediate ancestor. Shown here is an actual clonal lineage of the V1/V2-directed BnAbs CH01-CH04 (ref. 31). Targeting the unmutated ancestor with an immunogen that has enhanced binding may induce higher antibody responses²¹. If high-affinity ligands for unmutated ancestors cannot be found, then high-affinity ligands targeting the intermediate ancestors may be equally useful for triggering a response.