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Autoreactivity in an HIV-1 broadly reactive neutralizing antibody variable region heavy chain induces immunologic tolerance
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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 (VHDJH) 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 VHDJH 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 VHDJH 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 VHDJH 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.
The 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 possesses attributes often associated with autoreactivity: a heavy chain (HC) bearing an unusually long and hydrophobic third complementarity determining region (CDR3) (15, 16) that contains repeated arginine residues (17) and reactivity with various autoantigens, including cardiolipin, phosphatidylserine, and centromere and histone antigens (8, 10, 11). Other broadly neutralizing human HIV-1 mAbs (e.g., 4E10 and 1b12) (18, 19) also exhibit polyreactivity (8).
By generating a series of transgenic and/or knock-in mouse lines carrying an Ig HC variable region rearrangement (VHDJH) from a typical anti-DNA autoantibody, 3H9 (20–25), Weigert and colleagues demonstrated that the autoreactive properties of the 3H9 VHDJH were strongly penetrant, even in association with disparate light chains (LCs). One of the 3H9 knock-in mouse lines in particular, 3H9-76R (with the highest affinity for DNA within the 3H9 series), exhibited a profound developmental block at the pre-B to immature B cell transition (21, 25, 26) and elicited B cell tolerance in association with many LCs (24).
To determine whether the VHDJH properties of the 2F5 mAb might also be sufficiently autoreactive to elicit B cell tolerance regardless of LC pairing, we generated a knock-in mouse line, 2F5 VH, in which the VHDJH rearrangement of the original, human 2F5 mAb was targeted to the mouse Igh locus. This insertion allowed the robust development of large and small pre-B cells expressing chimeric human/mouse Igμ chains but resulted in a developmental blockade at the pre-B to immature B cell transition. This block significantly reduced peripheral B cell numbers; nonetheless, B220+ splenocytes in homozygous 2F5 VH knock-in mice contained similar frequencies of mature follicular B cells and underwent normal class switch recombination (CSR) to IgG that contained minimal reactivity to autoantigens. This developmental blockade in the bone marrow (BM) of 2F5 VH knock-in mice is nearly identical to that exhibited by 3H9-76R transgenic and knock-in mice (21, 25, 26) and demonstrates that whereas the chimeric 2F5 HC is capable of supporting murine B lymphopoiesis and maturation, the intrinsic autoreactive properties of the 2F5 HC are sufficient to trigger immunologic tolerance. Our results demonstrate that a neutralizing antibody for a viral disease is under the control of immunologic tolerance.
Human 2F5 VHDJH Rearrangement Forms Functional Chimeric Antibodies with Mouse CH.
We first tested in vitro whether mouse C regions impacted the association and binding properties of the original human IgG1 2F5 mAb (herein referred to as h2F5). To do this, we generated 2F5 VHDJH/mouse Cγ1 and 2F5 VκJκ/mouse Cκ expression constructs, cotransfected them into 293T cells, and assessed the 2F5 chimeric mouse/human recombinant antibody (m2F5) for its ability to bind lipid and mouse and human cell antigens. Indeed, m2F5 bound both gp41 and lipids comparably to h2F5 (Fig. 1 A and B). Moreover, m2F5, like h2F5, reacted with both human epithelial and mouse fibroblast nuclear antigens (Fig. 1 C and D) and neutralized HIV-1 (Fig. 1 legend and Table S1).
The chimeric, recombinant 2F5 antibody (m2F5) is functionally equivalent to the original human 2F5 mAb (h2F5) in vitro. (A and B) Functional comparison of m2F5 with h2F5 by SPR analysis demonstrating that antigen binding specificity and lipid reactivity are preserved in the recombinant m2F5 antibody. (A) m2F5 bound to the HIV-1 gp41 membrane-proximal external region (MPER) peptides gp41652–671 MPER (Top) and gp41656–683 MPER (Bottom), which include the 2F5 binding epitope (ELDKWAS). The binding of m2F5 (solid line) to MPER peptides was comparable to those of h2F5 (dashed line). The CD4i gp120 mAb 17b (dotted line) was used as a negative control. (B) Lipid reactivity was retained in m2F5 antibody, and binding to liposomes with either phosphatidylserine (dashed line) or cardiolipin (solid line) was observed with both h2F5 and m2F5 mAbs. (C) m2F5 and h2F5 antibodies react with comparable avidity and specificity to mouse NIH/3T3 cell nuclear antigens. (D) m2F5 and h2F5 antibodies have comparable reactivities to human HEp-2 cell nuclear antigens. In addition, m2F5 and h2F5 were tested in the TZM-bl HIV Env pseudovirus neutralization assay, and both antibodies neutralized HIV-1 strains B.BG1168, B.SF162, B.QH0692, B.JRFL, and C.92UG0237, but not C.TV-1. IC50 neutralization titer values for m2F5 and h2F5 ranged from 0.35 to 11.9 μg/mL and from 0.06 to 1.8 μg/mL, respectively (Table S1).
We also assessed the ability of chimeric 2F5 HCs to pair with mouse κ LCs in vitro by cotransfection of the 2F5 VHDJH/mouse Cγ1 expression construct with mouse κ LCs obtained from C57BL/6 splenic B cells by 5′ RACE PCR. To do this, four mouse κ LCs were arbitrarily selected to include the 4–52, 4–60, 4–70, and 9–96 V genes representing two Vκ families (Vκ4 and Vκ9) frequently used in the splenic C57BL/6 LC repertoire. In each case, cotransfections of the m2F5 HC resulted in the production of secreted, functional mAbs (Table S2). Significantly, of the four chimeric recombinant antibodies generated by these transfections, three exhibited cardiolipin polyreactivity as determined by surface plasmon resonance (SPR) and ELISA (Fig. S1).
Generation of 2F5 VH Knock-in Mice.
To determine whether the 2F5 mAb HC was sufficiently autoreactive to be regulated by immunologic tolerance, the original, somatically mutated 2F5 VHDJH rearrangement (13, 14) was knocked into the mouse Igh locus, replacing the JH1–4 region (Fig. 2). To confirm the expected homologous recombination event in the Igh locus, four independent ES cell clones were assessed for the predicted insertion (Fig. S2A), and heterozygote and homozygote offspring harboring germline transmission of the 2F5 VHDJH rearrangement (2F5 VH knock-in mice) were identified by PCR (Fig. S2B). 2F5 VH knock-in mice supported CSR to the endogenous Cγ1 locus in vivo (Fig. S2C) and thus provide a valid model for direct determination of whether the 2F5 VHDJH can induce B cell tolerance mechanisms.
Targeted replacement of the JH cluster with the 2F5 VH gene. Genomic structure of the 2F5 VH targeting construct, the endogenous Ig HC locus, the targeted allele after homologous recombination, and the targeted allele after Cre-mediated neo cassette deletion. The 2F5 VH expression cassette comprises a J558 H10 family VH promoter (p), an H10 split leader sequence (L), and the prerearranged 2F5 VHDJH segment (2F5 VH). Exons are represented as closed boxes, the Igh intronic enhancer (E) is represented by a circle, the 2F5 VH, neo, and CAG-DTA cassettes are represented by shaded boxes, and loxP sites are depicted as triangles. The indicated restriction fragment sizes are indicated for WT and targeted loci. The 5′ and 3′ probes used to verify homologous recombination events at the 5′ and 3′ regions of the JH-Eμ region, respectively, are shown as black bars. Also indicated are PCR primers to identify (i) homologous recombinant clones (black arrows), (ii) removal of the neo marker (gray arrows), and (iii) germline-transmitted heterozygous (2F5 VH+/−) and homozygous (2F5 VH+/+) mice (colored arrows). B, Bam HI; RV, EcoRV; n, Nde I.
Majority of B Cells Expressing 2F5 VH Are Deleted in the BM at the Pre-B to Immature B Cell Stage.
To examine the effect of the targeted 2F5 VHDJH insert at one or both Igh alleles on B cell development, we compared B cell ontogeny in BM of heterozygous (2F5 VH+/−) and homozygous (2F5 VH+/+) knock-in mice with that of C57BL/6 controls. Fractionation of total BM B cells from 2F5 VH+/− and 2F5 VH+/+ mice into pro-B/large pre-B (B220loCD43+), small pre-B (B220loCD43−), and immature/mature B (B220hiCD43−) fractions (27) demonstrated a profound reduction in surface Ig (sIg+) B cell subsets (B220hiCD43−), both in frequency (≈4-fold for both 2F5 VH+/− and 2F5 VH+/+ mice; Fig. 3) and absolute numbers (≈10-fold for both 2F5 VH+/− and 2F5 VH+/+ mice; Table S3). BM B cells were also labeled with antibodies specific for IgM and IgD to identify immature, transitional, and mature B cell populations. The frequency and absolute number of each population were also reduced in 2F5 VH mice, with the largest decreases observed in transitional B cell populations (≈7- or ≈20-fold reduced frequencies and ≈15- or ≈60-fold decreases in numbers in 2F5 VH+/− and 2F5 VH+/+ mice, respectively). These results demonstrated that 2F5 VH mice exhibited a major blockade in B cell development predominantly at the pre-B to immature B cell transition, which is consistent with the induction of tolerance by the deletion of immature B cells expressing the 2F5 Ig HC paired with many endogenous LCs. This developmental blockade at the immature B cell stage is similar to that previously reported for the autoreactive anti-DNA 3H9 knock-in mouse (21, 28) (Fig. S3 and Tables S4–S7).
Flow cytometric analysis of B cell development in the BM of C57BL/6 (WT), 2F5 VH+/−, and 2F5 VH+/+ mice. Left: Representative dot plot histograms, with numbers indicating the percentage of cells within total BM B cell populations (gated as singlet, live, lin− cells; lin = Ter-119, Gr-1, CD11b, CD4, and CD8). BM cells were isolated from 9–12-week-old female mice. Right: Statistical analysis of BM B cell subset frequencies with each black, open, and gray circle representing an individual WT, 2F5 VH+/−, and 2F5 VH+/+ mouse, respectively; horizontal lines represent averages for each group. Significance values were determined by a two-tailed Student's test: *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.0001; NS, not significant. Populations were defined as follows: pro/large pre-B (fractions A–C’; B220loCD43+), small pre-B (fraction D; B220loCD43−), immature B (B220int/loIgMloIgD−), transitional, T1+T2 (B220intIgMint/hiIgDlo), and BM mature (B220hiIgMintIgDhi).
2F5 VH+/− Knock-in Splenic B Cells Preferentially Express Endogenous HCs.
We suspected that if 2F5 HC-expressing B cells in heterozygote 2F5 VH mice escaped BM deletion, they should be counterselected in favor of B cells expressing endogenous Igh rearrangements, as in 3H9-76R mice (21). Thus, we examined surface expression of endogenous (IgMa) relative to 2F5 VH-targeted (IgMb) alleles in 2F5 VH+/− IgMa/IgMb F1 (F1) mice. Indeed, most IgM+ splenocytes from 2F5 VH+/− F1 mice expressed surface IgMa, indicating strong selection for the endogenous HC (Fig. 4A). This finding contrasts with IgM+ B cells in the BM, where allelic exclusion of the endogenous allele is largely maintained (Fig. 4B). As measured by the relative amount of IgMa and IgMb expression within the total IgM+ B cell fraction, we estimated the frequency of endogenous μHC expression in 2F5 VH+/− F1 mice to be ≈85% in the spleen and ≈40% in the BM (Fig. 4B). This preferential expression of the endogenous HC in splenic 2F5 VH+/− F1 cells suggests that 2F5 μHC-expressing peripheral B cell populations are either selected against or eliminate their 2F5 VH transgenes by intrachromosomal recombination, followed by rearrangement/expression of the alternate allele (22), although it cannot be formally ruled out that some of these cells may have undergone CSR. 2F5 μHC-expressing splenic B cells in 2F5 VH+/− F1 may have also down-modulated their B cell receptors (BCRs; Fig. 4A), a possibility that is consistent with the lower levels of IgM observed in cells that become anergized through receptor engagement (29, 30).
Flow cytometric analysis of WT and 2F5 VH transgenic HCs expressed on the surface of 2F5 VH+/− B cells. (A) Representative contour histograms of splenic B cell populations from WT IgHb/WT IgHa and 2F5 VH IgHb/WT IgHa F1 mice, with numbers indicating the percentage of IgMa+ and IgMb+ cells (bearing endogenous and 2F5 VH transgene-bearing HCs, respectively) within the singlet, live, total B (B220+CD19+) cell gate. (B) Frequencies of IgMa positive cells within IgM+ BM and splenic fractions shown for five and six mice per F1 group, respectively.
2F5 VH Knock-in Mice Have Severely Diminished Numbers of Mature Splenic B Cell Populations with Low Surface Ig Density.
Selection against 2F5 Ig HC+ BM B cells should lead to diminished numbers of peripheral B cells. Indeed, compared with littermate controls, the numbers of splenic B cells (B220+CD19+ lin−, live-gated) in 2F5 VH+/− and 2F5 VH+/+ mice were reduced by 72% and 86%, respectively (Table S8). To determine whether transitional, marginal zone (MZ), and mature B cell subset frequencies within this remnant splenic B cell population were altered, we stained 2F5 VH B220+ B cells with antibodies specific for CD23, CD93, and IgM (31). Interestingly, within this residual B cell population, the frequency of transitional IgMlo (T3) B cells was little changed, but transitional IgMhi (T1 and T2) subset frequencies were significantly reduced relative to normal controls in both 2F5 VH+/− and 2F5 VH+/+ mice (Fig. 5A and Fig. S4). Moreover, within the remaining total 2F5 VH+/+ splenic B cell population, normal frequencies of MZ B cells and follicular mature (B220+, CD93−, CD23+, and IgM+) B cells were observed, but relative to littermate controls, the latter displayed lower surface IgM densities. This pattern of decreased T1/T2 B cell frequencies, and relatively normal frequencies of mature B cell subsets (but with reduced membrane Ig levels) is also quite similar to that previously reported for the 3H9 knock-in mouse (21, 25, 28). The low surface IgM densities seen in both transitional and mature B cell splenic populations of 2F5 VH+/+ mice also mirror the low membrane IgM levels expressed by IgMb+ splenic B cells from 2F5 VH+/− F1 mice (Fig. 4A).
Flow cytometric analysis of splenic B cell development in C57BL/6 (WT), 2F5 VH+/−, and 2F5 VH+/+ mice. Representative dot plot histograms using the Allman classification scheme (32), with numbers in Top showing the number of total B cells (B220+) that were CD93 (AA4.1)+ transitional or CD93− (mature + MZ) B cells, and further gated to allow for enumeration of transitional T1–T3 populations within the B220+CD93+ fraction (Middle) or enumeration of MZ and mature B cell subsets within the B220+CD93− fraction (Bottom). Mean fluorescence intensities for staining of WT, 2F5 VH+/−, and 2F5 VH+/+ mature B cell subsets with PE-labeled anti-IgM were 756, 506, and 342, respectively.
2F5 VH Knock-in Mice Lack Serum Reactivity to Cardiolipin and Nuclear Autoantigens Despite Having Substantial Levels of Serum IgG.
2F5 VH+/− and 2F5 VH +/+ mice exhibited normal to elevated serum IgG levels relative to normal controls, respectively, but 2F5 VH+/+ mice alone expressed significantly lower levels of serum IgM (Fig. 6A). Despite the substantial levels of circulating serum Ig in 2F5 VH knock-in mice, sera from heterozygous and homozygous knock-in mice did not bind cardiolipin or nuclear autoantigens (Fig. 6B). This absence of reactivity is consistent with an autoantigen-specific blockade of B cell development and loss of autoreactive B cell populations in 2F5 VH knock-in mice.
Total or autoantigen-specific serum Ig levels and gp41 MPER-specific serum Ig or B cell reactivity in WT, 2F5 VH +/−, and 2F5 VH+/+ mice. (A) Total IgM and IgG serum antibody levels. Each dot represents an individual mouse; horizontal lines represent mean serum antibody levels. Significance values were determined by a two-tailed Student's test: *P ≤ 0.05; ***P ≤ 0.0001; NS, not significant. (B) Total serum Ig reactivity against plate-bound 2F5 nominal epitope (gp41652–671 MPER) peptide, nuclear antigens, and cardiolipin. For gp41 MPER, ANA, and cardiolipin reactivity assays, MRL/lpr serum was used as a positive control. Each dot represents an individual mouse; horizontal lines represent mean serum antibody levels.
The development of a safe and effective HIV-1 vaccine has been blocked by the inability to design HIV-1 immunogens that induce antibodies that potently neutralize diverse HIV-1 strains. Although the HIV-1 Env has conserved regions to which rare, broadly neutralizing human antibodies bind, either on immunogens or in the context of natural infections, these conserved regions only rarely induce broadly neutralizing antibodies (6, 7, 32). Moreover, even on the rare occasions that broadly neutralizing antibodies are induced by HIV-1 infection, they only arise months after infection (33).
That the 2F5 VH knock-in mouse shows a profound block in expression of the 2F5 VH at the immature B cell stage demonstrates that the 2F5 VH is sufficiently autoreactive to invoke tolerance control of 2F5 VH expression, and supports the notion that expression of this specificity is regulated by tolerance mechanisms in vivo. In this regard, many of the broadly neutralizing antibodies, such as mAbs 4E10 and 1B12, share some characteristics of the 2F5 HC, including long hydrophobic CDR3s and polyreactivity, characteristics previously associated with antibodies marked for deletion in human BM (34).
It is possible that the profound block in B cells expressing 2F5 VH-containing HCs may be enhanced by incomplete and/or inefficient pairing of chimeric 2F5 VH/mouse CH HCs with endogenous mouse LCs. Our data, however, do not support this possibility. First, the relatively normal pre-B compartment in 2F5 VH knock-in mice (comparable to that in the 3H9 knock-in mouse; Tables S4–S7) is most consistent with the ability of the 2F5 μHC to associate efficiently with surrogate LC and support continued differentiation to the immature B cell stage, although this could also be due to compensation of an earlier, partial pre-B cell defect by autoreactive, immature B cells arrested at the pre-B cell stage. Either possibility, however, is consistent with the ability of the 2F5 μHC to form signaling-competent BCR and/or pre-BCR complexes rather than pairing incompatibility. Second, the cotransfection of the m2F5 HC with four distinct mouse LCs produced functional recombinant antibodies that reacted with self-antigens (Fig. S1). This observation demonstrates the capacity for LC pairing and the substantial penetrance of the 2F5 autoreactive phenotype. Third, we generated 26 hybridoma lines from the spleens of naïve 2F5 VH knock-in mice containing the 2F5 μHC in association with κLCs using 9 different Vκ gene families (Table S9). Fourth, 2F5 VH knock-in mice exhibit normal ratios of MZ B cells and follicular B cells (Fig. 5), despite significant reductions in splenic B cell numbers (Table S8). This observation indicates that the capacity for normal B cell maturation is retained. Finally, it could be argued that the lack of serum IgM in 2F5 VH+/+ mice is due to the 2F5 HCs inability to pair with LCs similar to the phenotype reported in κ+λ LC- deficient mice, which make no serum IgM but have detectable serum IgG composed of HC dimers (35). However, 2F5 VH+/+ knock-in mice have substantial levels of serum IgG/κ, as demonstrated by the use of anti-κLC Ab to capture serum IgG for ELISA quantification. Taken together, these observations strongly suggest that immunologic tolerance, not impaired HC+LC pairing, is the most likely explanation for reduced B cell numbers in 2F5 VH knock-in mice.
The peripheral phenotype in 2F5 VH+/+ mice is consistent with additional mechanisms for controlling autoreactivity in residual splenic 2F5 VH-bearing B cells that have escaped central tolerance. In particular, the relative enrichment for the T3 IgMlo population in 2F5 VH+/+ mice (Fig. 5) is consistent with increased frequencies of autoreactive B cells that become anergized through receptor engagement (29, 30). A similar IgMlo phenotype has also been described in splenic transitional B cells in the various 3H9 mouse lines (21, 23, 25, 26, 28), reflecting frequent anergic B cells, or alternatively, cells that have undergone LC editing (28, 36, 37). Interestingly, 2F5 VH+/+ mature B cells also exhibit lower sIgM densities, similar to anergic anti-Sm transgenic mature B cell fractions (38). An intriguing alternative explanation for reduced sIgM expression in mature 2F5 VH+/+ B cell populations is that their autoreactive BCRs bind to an intracellular antigen, analogous to mature B cell populations in the hen egg lysozyme model (39). Regardless of the reason for the lowered IgM levels in mature 2F5 VH+/+ B cells, the fact that such populations are present at normal ratios, coupled with the abundance of nonautoreactive serum Igs in 2F5 VH+/+ mice, predicts that additional mechanisms (other than anergy) purge autoreactivity in these populations. In various 3H9 knock-in lines, such additional mechanisms of tolerizing 3H9-bearing dsDNA-reactive mature B cells include LC receptor editing, or replacement of the 3H9 insert by a secondary VH→VHDJH rearrangement (i.e., VH replacement) (20, 24, 40). It will be critical to determine which of these peripheral B cell tolerance mechanisms and/or anergy operate in 2F5 VH+/+ mice.
Mice bearing conventional or targeted autoreactive Ig transgenes have been critical in defining the developmental stages in which self-reactive B cells are eliminated (41). The 2F5 VHDJH knock-in mouse line demonstrates that the great majority of B-lineage cells that express the 2F5 VHDJH rearrangement are halted in their development at the transition from small pre-B to immature B cells (Fig. 3). This developmental blockade is nearly identical to that observed in mice that express the 3H9-76R VHDJH rearrangement that specifies anti-DNA reactivity in association with many LCs (24). If the germline 2F5 VHDJH rearrangement does not specify autoreactivity, immature B cells carrying the 2F5 HC would not be tolerized and the 2F5 HC CDR residues critical for self-reactivity and HIV-1 neutralization must have arisen in germinal centers. Significantly, removal of autoreactive B cells can also occur in germinal centers (42), and it is possible that 2F5 HC B cells carrying somatically generated mutations critical for self-reactivity/HIV-1 neutralization may normally be deleted or modified during the germinal center reaction. Future studies to determine whether tolerance mechanisms act similarly on the 2F5 germline VHDJH sequence will be informative and complementary to these studies.
Both the 2F5 and 4E10 mAbs bind to the gp41 membrane proximal region on HIV-1 virions, as well as to the lipid bilayer (10). Mutation of hydrophobic residues in the 2F5 HC CDR3 abrogates both lipid binding and neutralization of HIV-1 (43). The induction of neutralizing antibodies specific for this region will likely require the targeting of B cell populations that can make antibodies that bind both lipids and gp41 Env epitopes. This requirement may be facilitated by the activation of dendritic cells or other antigen-presenting cells capable of promoting vaccine-induced B cell responses that normally do not occur. The 2F5 mAb has been safely administered to a number of humans, and 2F5 does not have characteristics of a pathogenic lipid autoantibody (i.e., it does not require β-2-glycoprotein-1 to bind to lipids) (8, 44, 45). However, if these antibodies can be induced, safety monitoring in nonhuman primate trials will be of paramount importance.
Our studies demonstrate that the HIV-1 broadly neutralizing antibody 2F5-containing HC is sufficiently autoreactive to trigger immunologic tolerance in the setting of a knock-in mouse. Our findings have important implications for the design of strategies to induce neutralizing antibodies to the HIV-1 Env gp41 membrane-proximal external region (MPER). HIV-1 vaccine development should focus on vaccine regimens that might safely circumvent these tolerance controls. Moreover, efforts should concentrate on accelerating and broadening those neutralizing antibody responses that are readily made in response to HIV-1, such as autologous neutralizing antibodies that arise months after natural HIV-1 infection (46, 47).
Expression/Characterization of m2F5 and Generation of 2F5 VH Mice.
The methods and reagents used to generate m2F5 and the binding, immunofluorescence, and neutralization assays used to characterize its functional properties are described in SI Methods, as are the reagents and methods used for the site-directed targeting of 2F5 VH into the mouse Igh locus.
Mice and Flow Cytometry.
Female C57BL/6 and C57BL/6 Igha, inbred mouse strains (8–12 weeks of age) were purchased from Charles River Laboratories. 3H9 mice, originally produced in the laboratory of Dr. Martin Weigert on a BALB/c background, were backcrossed onto the C57BL/6 background for >14 generations in the laboratory of Dr. Robert Eisenberg (University of Pennsylvania).
For flow cytometric analysis, BM cells and splenocytes were isolated from 9–12-week-old female mice. Total BM B cells (gated as singlet, live, CD19+, lin− lymphocytes; lin = Ter-119, Gr-1, CD11b, CD4, and CD8) were stained with APC anti-B220 and phycoerythrin (PE) anti-CD43 antibodies or FITC anti-IgD and PE anti-IgM antibodies; singlet, live, lymphocyte-gated splenocytes were stained using the combination of FITC anti-B220, PE anti-IgM, APC anti-CD93, and PE-Cy7 anti-CD23 antibodies. Data were acquired using a BD LSR II flow cytometer equipped with FACS Diva software and analyzed using FloJo software.
Allotype Screening.
2F5 VH IgHb/WT IgHa and WT IgHb/WT IgHa F1 mice were generated by breeding C57BL/6 Igha congenic mice with 2F5 VH+/− mice and WT littermate controls, respectively. BM cells and splenocytes from 8–16-week-old female F1 mice from each group were surface stained with PE-IgMa and FITC-IgMb antibodies, distinguishing targeted 2F5 VH μHCs bearing the allotype of the targeted IgH allele (IgMb) from endogenous μHCs bearing the IgMb allotype.
ELISA Analyses.
Serum samples were collected from naïve female WT, 2F5 VH+/−, and 2F5 VH+/+, and where applicable, MRL/lpr mice. Serum concentrations of total IgG and IgM were determined using quantitative mouse IgG and IgM ELISA kits, respectively (Bethyl). ELISA measurements of cardiolipin and gp41 MPER 2F5 reactivity of total (IgM+IgG-specific) Igs was determined by optical density readings, as previously described (8, 10), and serum reactivity of total Igs to nuclear autoantigens was determined using a mouse anti-ANA quantitative ELISA kit (Alpha Diagnostics). Cardiolipin and ANA assays were done using serum from 12–32-week-old mice; all other assays were done using serum from 8–16-week-old mice.
We thank John Whitesides, Patti McDermott, and Letealia Oliver for expert technical assistance in flow cytometry; Robert Parks for ELISA assays; and Richard Scearce, Jennifer Hutchinson, and Heather Stevenson for assistance with cloning and expression of recombinant antibodies. 3H9 mice on the C57BL/6 background were provided by Dr. Robert Eisenberg (University of Pennsylvania). This study was supported by a Collaboration for AIDS Vaccine Discovery Grant from the Bill and Melinda Gates Foundation (to B.F.H., G.K., and L.V.), an Innovation Grant from the Duke Center For AIDS Research (to L.V.), and by National Institutes of Health, National Institute of Allergy and Infectious Diseases, Division of AIDS, Center for HIV/AIDS Vaccine Immunology Grant AI0678501 (to G.K. and B.F.H.).
The authors declare no conflict of interest.
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Funding
Funders who supported this work.
NIAID NIH HHS (2)
Grant ID: R01 AI087202
Grant ID: AI0678501