We previously reported that some of the rare broadly reactive, HIV-1 neutralizing antibodies are polyreactive, leading to the hypothesis that induction of these types of neutralizing antibody may be limited by immunologic tolerance. However, the notion that such antibodies are sufficiently autoreactive to trigger B cell tolerance is controversial. To test directly whether rare neutralizing HIV-1 antibodies can activate immunologic tolerance mechanisms, we generated a knock-in mouse in which the Ig heavy chain (HC) variable region rearrangement (V H DJ H ) from the polyreactive and broadly neutralizing human monoclonal antibody 2F5 was targeted into the mouse Igh locus. In vitro, this insertion resulted in chimeric human/mouse 2F5 antibodies that were functionally similar to the human 2F5 antibody, including comparable reactivity to human and murine self-antigens. In vivo, the 2F5 V H DJ H insertion supported development of large-and small pre-B cells that expressed the chimeric human/mouse Igμ chain but not the production of immature B cells expressing membrane IgM. The developmental arrest exhibited in 2F5 V H DJ H knock-in mice is characteristic of other knock-in strains that express the Ig HC variable region of autoreactive antibodies and is consistent with the loss of immature B cells bearing 2F5 chimeric antibodies to central tolerance mechanisms. Moreover, homozygous 2F5 V H DJ H knock-in mice support reduced numbers of residual splenic B cells with low surface IgM density, severely diminished serum IgM levels, but normal to elevated quantities of serum IgGs that did not react with autoantigens. These features are consistent with elimination of 2F5 HC autoreactivity by additional negative selection mechanism(s) in the periphery.2F5 | broadly neutralizing antibodies | B cell development | autoantigens T he development of a safe and effective vaccine for HIV-1 is a global priority. Although anti-HIV-1 CD8 T cell responses can help control the level of viral load (1), they alone do not prevent infection (2). In contrast, administration of human mAbs targeted to conserved regions of the HIV-1 envelope (Env) in nonhuman primates, before challenge with simian-HIV (SHIV) viruses, can protect against infection (3-5). However, a major obstacle preventing development of an effective HIV vaccine is the inability to induce broadly reactive neutralizing antibodies routinely (6, 7).Several hypotheses have been offered to explain the absence of effective vaccine-induced immune responses to conserved, neutralizing epitopes of the HIV-1 Env, including suppression of neutralizing antibody responses by immunologic tolerance (8, 9). This hypothesis arose from the observation that many broadly reactive neutralizing HIV-1 antibodies also react with a variety of self-antigens (8-11). This hypothesis, however, is controversial because the rare, neutralizing human mAb 2F5 reacts with low affinity to autoantigens (8-12). mAb 2F5 was derived from an HIV-1 infected subject (13, 14) and protects against SHIV challenge (5). mAb 2F5 pos...

The HIV-1 broad neutralizing antibody (bnAb) 2F5 has been shown to be poly/self-reactive in vitro, and we previously demonstrated that targeted expression of its VDJ rearrangement alone was sufficient to trigger a profound B cell developmental blockade in 2F5 VH knockin (KI) mice, consistent with central deletion of 2F5 H chain-expressing B cells. Here, we generate a strain expressing the entire 2F5 bnAb specificity, 2F5 VHxVL KI mice, and find an even higher degree of tolerance control than observed in the 2F5 VH KI strain. Although B-cell development was severely impaired in 2F5 VHxVL KI animals, we demonstrate rescue of their B-cells when cultured in IL-7/BAFF. Intriguingly, even under these conditions, most rescued B-cell hybridomas produced mAbs that lacked HIV-1 Envelope (Env) reactivity due to editing of the 2F5 L chain, and the majority of rescued B-cells retained an anergic phenotype. Thus, when clonal deletion is circumvented, κ editing and anergy are additional safeguards preventing 2F5 VH/VL expression by immature/transitional B-cells. Importantly, 7% of rescued B-cells retained 2F5 VH/VL-expression and secreted Env-specific mAbs with HIV-1 neutralizing activity. This “partial” rescue was further corroborated in vivo, as reflected by the anergic phenotype of most rescued B-cells in 2F5 VHxVL KI × Eμ-bcl2 tg mice, and significant (yet modest) enrichment of Env-specific B-cells and serum Igs. The rescued 2F5 mAb-producing B-cell clones in this study are the first examples of in vivo-derived bone marrow precursors specifying HIV-1 bnAbs, and provide a starting point for design of strategies aimed at rescuing such B-cells.

A feature of Ig hypermutation is the presence of hypermutable DNA sequences that are preferentially found in the V regions of Ig genes. Among these, RGYW/WRCY is the most pronounced motif (G:C is a mutable position; R = A/G, Y = C/T, and W = A/T). However, a molecular basis for the high mutability of RGYW was not known until recently. The discovery that activation-induced cytidine deaminase targets the DNA encoding V regions, has enabled the analysis of its targeting properties when expressed outside of the context of hypermutation. We analyzed these data and found evidence that activation-induced cytidine deaminase is the major source of the RGYW mutable motif, but with a new twist: DGYW/WRCH (G:C is the mutable position; D = A/G/T, H = T/C/A) is a better descriptor of the Ig mutation hotspot than RGYW/WRCY. We also found evidence that a DNA repair enzyme may play a role in modifying the sequence of hypermutation hotspots.