Broadly neutralizing Abs against HIV protect from infection, but their routine elicitation by vaccination has not been achieved. To generate small animal models to test vaccine candidates, we have generated targeted transgenic (“knock-in”) mice expressing, in the physiological Ig H and L chain loci, two well-studied broadly neutralizing Abs: 4E10, which interacts with the membrane proximal external region of gp41, and b12, which binds to the CD4 binding site on gp120. 4E10HL mice are described in the companion article (Doyle-Cooper et al., J. Immunol. 191: 3186–3191). In this article, we describe b12 mice. B cells in b12HL mice, in contrast to the case in 4E10 mice, were abundant and essentially monoclonal, retaining the b12 specificity. In cell culture, b12HL B cells responded avidly to HIV envelope gp140 trimers and to BCR ligands. Upon transfer to wild-type recipients, b12HL B cells responded robustly to vaccination with gp140 trimers. Vaccinated b12H mice, although generating abundant precursors and Abs with affinity for Env, were unable to rapidly generate neutralizing Abs, highlighting the importance of developing Ag forms that better focus responses to neutralizing epitopes. The b12HL and b12H mice should be useful in optimizing HIV vaccine candidates to elicit a neutralizing response while avoiding nonprotective specificities.
Broadly neutralizing Abs (bNAbs) to HIV recognize relatively conserved sites on HIV envelope (Env) protein (gp120/gp41) that are similar among many isolates and clades (reviewed in Refs. 1–3). The bNAbs to HIV arise in ≤ 25% of patients after many months of infection (4), although only ∼ 1% are considered “elite neutralizers” (5). However, single bNAbs are not generally protective in the context of chronic HIV infection. High viral loads and the mutability of HIV lead to escape mutants. Nevertheless, because passive transfer of several bNAbs can prevent infection in animal models, bNAbs are predicted to be protective if elicited prior to infection (2). Therefore, the formulation of vaccines capable of eliciting bNAbs is a high priority in efforts to prevent infection. In this article, we focus on bNAb b12, which was until recently one of the most potent and broadly neutralizing HIV Abs known.
The bNAb b12 was initially identified as one of a group of phage-displayed Abs generated from bone marrow (BM) RNA of an asymptomatic HIV patient who had been infected for 6 y (6). Among 32 gp120-binding phages, b12 belonged to a cohort of 4 sharing CDR3 regions in both H and L chains. b12 was back-engineered to encode a full IgG molecule. Its crystal structure was striking because the putative Ag combining site was marked by extreme protrusion of the H chain CDR3 (7). The structure was interpreted to indicate that the H chain CDR3 might make major contacts with the CD4 binding site, consistent with H and L chain shuffling experiments, which indicated that the b12 H chain retained specificity when paired with many different L chains. However, alteration of four specific somatically mutated residues in CDR1 and CDR3 of the b12 L chain abolished binding to gp120, suggesting that the L chain contributes contacts (8). By contrast, the b12 L chain, when paired with random H chains from the same library and selected for gp120 binding, reselected the same H chain CDR3 (9). The b12 Fab was subsequently cocrystallized with a truncated, disulfide-stabilized core of gp120, revealing a structure in which all contacts were with H chain (10).
Surprisingly, in addition to its ability to bind to HIV Env, b12 has been suggested to be an autoantibody (11). This conclusion was based mainly on Ab binding studies. As we discuss in the companion article (12), this claim was extended to the gp41 Abs 4E10 and 2F5. b12 was found to bind to ribonucleoprotein, dsDNA, centromere protein, histones, and HEp-2 cells in a cytoplasmic and nucleolar pattern (11). These data raised the possibility that conserved HIV epitopes might evade the immune system by mimicking self and thereby provoking clonal elimination of reactive B cells. The recessed CD4 binding site might tend to require unusually long, extended CDRH3 regions for Ab neutralization. Long CDRH3s have been associated with polyreactivity/autoreactivity (13–15), and might be counterselected by tolerance. However, thus far, the self-reactivity ascribed to b12 is solely based on Ab binding assays, which are subject to a number of technical caveats and do not necessarily correlate with in vivo reactivity or tolerance.
In this article, we present data on the generation and analysis of knock-in mice in which the variable portions of b12 were introduced to the physiological mouse Ig H and L loci by gene targeting. We find that B cells in these mice retain the transgene-encoded specificity, and appear not to be negatively regulated by immune tolerance. The b12 H-only and H/L mice should prove useful in assessing vaccine candidates for the ability to drive B cell responses.
Materials and Methods
H chain constructs were targeted in the C57BL/6-derived C2 embryonic stem (ES) cell line (16) at The Scripps Research Institute Mouse Genetics Core Facility. The L chain construct was targeted in C57BL/6-derived ES cells by inGenious Targeting Laboratories (Ronkonkoma, NY). Briefly, targeting replaced the DQ52-JH cluster in the IgH locus with a b12 H chain VDJ exon, flanked on the 5′ side by a loxP flanked neo gene, and the mouse VHJ558.85.191 promoter, leader, and intron. Similarly, b12 L chain VJ element was targeted to replace the Jκ cluster. The L chain gene targeting vector was pVKR2neo, and L chain targeting was carried out essentially as described (17). The L chain VJ sequences were fused on the 5′ end with a promoter, leader, and intronic sequence from mouse Vκ4-53 and introduced as a NotI–SalI fragment. The targeting plasmid was then linearized with ClaI.
For H chain targeting, we modified the pEASY-FLIRT vector (18). A 3.9-kb 5′ homology arm was cloned into the NotI site, and a 2.6 kb 3′ homology arm was introduced as an AscI–MluI fragment into the AscI site. A 5′ 1540-bp VHJ558.85.191 promoter region, including leader exon and intron, was generated. The b12 VDJ was then “sewn” onto the VH promoter, using overlapping extension PCR with primers carrying MluI sites. The promoter–VDJ was then cloned into the AscI site of the targeting vector. Plasmid was linearized using an SfiI site 5′ to the upstream arm of homology and used for electroporation of ES cells, followed by G418 selection. ES cells carrying the desired targetings were identified through a series of PCR reactions carried out with genomic DNA from ES cell clones and using Phire polymerase (Thermo), under the recommended conditions and annealing temperatures listed below. Primer locations are shown in Supplemental Figs. 1 and 2 of the accompanying paper. L chain targeting screening analysis was with primers L1 (5′-CAAGTAGACCTCTCTAATCTTGGTTAG-3′) and L2 (5′-TTCTATCGCCTTCTTGACGA-3′), with 58°C annealing temperature. Candidate clones were further identified by a PCR using primers L6 (5′-CCAAGGAGGGATCATGTGTTTGAAT-3′ and L7 (5′-TCAGTGTCACAGGTAGAGATGGGT-3′), with 63°C annealing temperature.
H chain targetings were identified using primers H1 (5′-AGGTAAGGAGCTCAGCAGTTCCAA-3′) and H2 (5′-GCCAGCCTAGTTTAGCTTAGCGGCCCA-3′), with 63.5°C annealing temperature. Candidate clones were confirmed by a PCR using primers H7 (5′-AGGAGTATTCCTGTTCTCCCATGCCTGCATATCA-3′) and H6 (5′-GCTAAAGCGCATGCTCCAGACTG-3′), with 63.5°C annealing temperature. PCR products from ES cells chosen for mouse generation were verified by sequencing. Neor genes were eliminated in the germline by breeding to EIIa-cre–expressing mice. Targetings were confirmed by Southern blotting using SacI or BamH1 digestion and a 2-kb EcoRI 5′ probe in the case of the L chain gene or BamH1 digestion and a 0.5-kb Xma–BamH1 3′ probe in the case of the H chain gene.
All experiments were performed in accordance with relevant institutional and national guidelines and were approved by The Scripps Research Institute Institutional Animal Care and Use Committee. C57BL/6J, EIIa-cre, and IgHa mice on the B6 background (B6.Cg-Igha Thy1a Gpi1a/J) were from The Jackson Laboratory.
Flow cytometry analyses
Analyses for surface markers and Ca2+ flux assay were performed using standard protocols, as previously described (1920) followed by Streptavidin-PE or Streptavidin-APC. Samples were read on an LSR II instrument (BD) and analyzed using the FlowJo program (TreeStar).
YU2 gp120-His6 AviTag, and YU2 gp140-F-His6 or JRFL gp140-F-His6 AviTag, were prepared as previously described (21). The uncleaved JRFL full-length gene used in Fig. 6H carried a pair of R-to-S mutations in the REKR furin cleavage site, which was also a feature of the soluble gp140 trimers used. Supernatants were purified by Galanthus nivalis lectin chromatography, followed by size exclusion chromatography.
Lymphocytes from b12H or b12HL spleens (SPs) were cultured for 3 d in Advanced-DMEM supplemented with 10% FCS, 20 mM GlutaMax, 55pM 2-ME, IL-4 (20 ng/ml), and anti-CD40 (10 μg/ml) and fused with the SP2/0 myeloma line, using polyethylene glycol. Cells from each fusion were then distributed into 96-well plates and hybrids selected with hypoxanthine/aminopterin/thymidine medium. Env-reactive hybrids were selected by ELISA screening of culture supernatants against JRFL gp140.
Neutralization activity of Abs against pseudovirus in TZM-bl cells was determined as described previously (22, 23). Briefly, TZM-bl cells were seeded in a 96-well flat-bottom plate and infected with pseudovirus in the presence of inhibitors (200 μl, total volume). Viruses were preincubated with the Ab for 1 h at 37°C. Luciferase reporter gene expression was quantified 72 h post infection upon lysis and the addition of luciferase substrate (Promega).
Statistical analyses were performed using Prism (GraphPad). Differences between two groups were assessed by the two-tailed t test; differences between three or more groups were evaluated by one-way ANOVA, followed by the Bonferroni multiple comparison posttest.
Generation of b12 H and L knock-in mice
C57BL/6 ES cells were modified by gene targeting to introduce HIV Ab H and L chain variable exons to the natural loci, replacing the respective J clusters, essentially following the strategies described (17, 24–26). Appropriate gene targeting and neo gene removal were carried out as described in Materials and Methods; targeting verification was as described in the supplemental figures of the accompanying manuscript. Mice carrying modified H and L loci of b12 were interbred. We then analyzed B cell development and HIV Ag binding by B cells in mice carrying targetings of H or L, or both, Ig loci.
B cells of b12H and b12HL mice are abundant and bind to soluble Env
We first compared mice carrying one or both b12 Ig chains. Mice carrying only the H chain gene (b12H) were bred to IgHa/a mice, and expression of the endogenous and transgenic alleles on B cells was measured by flow cytometry. The b12H mice displayed excellent allelic exclusion, with > 95% of B cells expressing only the “b” allele, indicating successful expression and feedback suppression of endogenous rearrangements (Fig. 1A, 1B). The b12H mice had normal numbers of B lymphocytes in the BM, SP, and lymph nodes (LNs), although immature B cells in BM were reduced (Fig. 2A, 2B) and B cell numbers in LN were higher than in controls (Fig. 2C). Mice carrying both H and L genes (b12HL) had B cell numbers similar to those in b12H mice (Fig. 2B, 2C).
We next assessed B cells able to bind HIV Ag. A significant subset (∼30%) of b12H B cells and > 90% of all mature b12HL B cells could bind to soluble trimers of HIV Env (the JRFL isolate), as measured by flow cytometry (Fig. 2D). Absolute numbers of B cells falling into the JRFL+ gate are shown in Fig. 2E. These data indicated that a significant subset of endogenous L chains is permissive for Env binding when paired with b12H and that in b12HL B cells the transgenic L chain was paired with b12 H chain.
Analysis of sera from naive mice revealed that b12H and b12HL mice had normal IgG levels and slightly reduced IgM levels (Fig. 3A, 3B). b12HL sera, but not b12H sera, contained high titers of HIV Env IgG binding activity (Fig. 3C), which was able to neutralize JRFL (Fig. 3D). As we show in a later section, the low background levels of anti-HIV activity of b12H, compared with b12HL, preimmune sera were likely the result of both the lower frequency and the lower average affinity of B cells reactive to Env. The ability of b12HL B cells to undergo H chain class switch yet retain b12 specificity supported the idea that the H chain transgene was appropriately targeted.
Maturity and functionality of peripheral B cells
Analysis of B cell maturation revealed that Env-binding B cells in SP of b12H and b12HL mice had mostly a mature follicular phenotype, expressing CD21 and CD23 (Fig. 4A) and mainly lacking expression of CD93 (Fig. 4B), a marker expressed by immature and anergic B cells (27–29). A significant subset of JRFL-binding cells had a marginal zone (MZ) B cell phenotype (CD21highCD23intermediate, Fig. 4A). MZ B cells respond rapidly to bloodborne microbes, whereas follicular B cells play a larger role in high-affinity secondary responses (30). Furthermore, splenic B cells from b12HL and b12H mice gave normal BCR-triggered Ca2+ responses and, unlike wild-type (WT) B cells, could respond to soluble HIV Env trimers, with the b12HL response being particularly robust (Fig. 4C). Because b12HL B cells showed normal development, were abundant, recognized HIV Ags, triggered normally, and were able to produce high levels of b12 IgG in serum, we conclude that the b12 BCR is “innocuous—that is, not autoreactive to a physiologically significant extent.
Analysis of b12HL B cells in mixed chimeras
Because B cell monoclonality can rescue survival and function of anergic B cells owing to reduced B cell:B cell competition (31, 32), we tested whether b12HL B cells competed well with normal B cells in mixed BM chimeras. The b12HL and WT BMs were mixed at a 1:1 ratio and used to reconstitute Rag1−/− mice. At 6 wk after reconstitution, PBLs were analyzed by flow cytometry, revealing an average of 10% of B cells able to bind Env gp120 (YU2) (Fig. 5A). This reduced contribution to the B cell compartment relative to the BM proportion is typical of “innocuous” BCR transgenics, owing to accelerated B cell development at the progenitor stage (32, 33). These cells had CD93 levels no higher than did wild-type B cells, indicating that they were neither anergic nor immature (Fig. 5B), and the frequency of CD93+ cells was no higher (11.7 ± 2.4% versus 16.7 ± 3.5%; n = 8; p = 0.2492). The ability of these B cells to respond to immunization was tested by challenging chimeras 6 wk after reconstitution with soluble JRFL gp140 trimers in adjuvant, followed by boosting 3 wk later. The chimeras could rapidly make significant levels of IgG and IgM after boosting that cross-reacted to a heterologous Env isolate, YU2 (Fig. 5C, 5D). It is not clear why the primary IgM response was low in the immunized mice. As WT mice failed to respond rapidly to this challenge, presumably because the precursor frequency of reactive B cells is too low, we infer that the responses came from the b12HL B cells. We conclude that b12HL B cells compete well with WT cells and can respond to HIV candidate vaccines.
b12H B cells bind to gp140 but do not efficiently neutralize HIV
As normal sera of b12H mice failed to neutralize JRFL pseudovirus, we tested whether it was possible to elicit these Abs by immunization. The b12H and WT mice were challenged with soluble JRFL trimers in Ribi adjuvant, then boosted 3 wk later. Although b12H mice produced both IgM and IgG JRFL-reactive Ab (Fig. 6B, 6C), the IgG response was no better than in WT. By contrast, an IgG component cross-reactive to YU2 or to the synthetic Env mimetic scaffold 2bodx_43 (34) was rapidly elicited in b12H mice (Fig. 6D, 6E) but not in WT mice, although at 4 wk a response arose to YU2 in WT mice. (Fig. 6A demonstrates that 2bodx_43 is recognized by a large proportion of preimmune b12H B cells.) These data indicated that the WT response was not directed to the CD4 binding site of Env. Assays of neutralizing activity indicated that immunized b12H mice made a small but significant JRFL-neutralizing response, although much weaker than that present in unmanipulated b12HL mice. The weak, but detectable, response of b12H mice suggests that this model might facilitate a comparison of immunogens designed to elicit a neutralizing response while avoiding production of Abs to “distracting” epitopes that are nonprotective.
To understand why neutralization activity in sera of b12H mice was weak when gp140-binding B cells were numerous, we studied the specificity of preimmune b12H B cells in more detail. IgG1-producing hybridomas were generated from b12H SP cells activated in vitro with IL-4 and anti-CD40. As expected, many hybrids produced Abs reactive to gp140. Their relative binding avidity to JRFL gp140 trimers varied but could approach that of b12HL Ab (Fig. 6G, “b12”). However, when tested for their ability to bind to cleaved (natural JRFL) or uncleaved JRFLΔCT trimers expressed as full-length protein on 293T cells, the b12H hybrids tested bound poorly to cleaved compared with uncleaved versions of Env (Fig. 6H). The b12, by contrast, bound much better to cleaved trimers. These data confirm that the b12 L chain not only makes a critical contribution to binding affinity but also is required for neutralization.
We generated knock-in mice expressing broadly neutralizing HIV Abs and analyzed their B cell development to determine if and how B cells carrying these specificities are negatively regulated by immune tolerance processes. The b12HL B cells appear not to be negatively selected, as judged by their robust generation, lack of receptor editing, maintenance of b12 specificity, and ability to be triggered by HIV Ags and to secrete Ab. Similarly, b12H B cells are present in large numbers and many bind HIV Env, suggesting that a large fraction of endogenous L chains are permissive for binding. This idea is consistent with crystal structure and chain swapping data indicating that the H chain is largely responsible for b12 specificity (8–10).
The suggestion that b12 might be autoreactive (11) was not supported by our studies. Not only could these cells be triggered in vitro by HIV ligands, but also they responded robustly to soluble gp140 trimers in vivo. By contrast, as we show in the accompanying paper, 4E10 appears to be autoreactive and regulated by tolerance. Our findings illustrate the importance of bioassays to identify physiologically significant autoreactivity.
The b12H chain has a long CDR3 region. Long CDRH3s have been documented in some studies to promote polyreactivity, and to present a barrier to B cell development owing to problems with immune tolerance, pairing with surrogate L chain components at the precursor stage and, possibly, pairing with L chains at the small pre-B stage (13, 14, 35–38). It is therefore significant that the b12H mouse demonstrates no evidence of B cell developmental defects. B cells carrying the transgene specificity were abundant in the peripheral lymphoid organs and apparently fully functional.
Because of the functionality of their Env-reactive B cells, b12H and b12HL mice should be useful models for HIV vaccine development. In vitro assays can be carried out with primary Env-reactive B cells. Many B cells produced by these mice recognize Env, allowing one to easily monitor ongoing responses, to rank the efficacy of vaccine candidates, and to detect weak or even abortive responses.
The results from immunization of b12H mice with experimental vaccine candidates illustrate the daunting challenge of HIV vaccination and might contribute to our understanding of how best to formulate and design vaccines. The b12H mice contain a huge number of B cells with Env reactivity but lacking in neutralizing capacity. We presume that a very small subset of these cells might have appropriate L chains that promote or permit eventual neutralizing capacity either prior to or after appropriate somatic mutation. Cleaved Env trimers on the surface of virions are required for infection and are the targets of neutralizing Abs such as b12. But randomly selected B cells from b12H mice captured as hybridomas preferentially recognize the uncleaved form. This result emphasizes the importance of cleavage for the generation of a conformation of Env trimer accurately mimicking that of the functional trimer on the virion surface or on transfected/infected cells (39). The only recombinant Env trimers that are cleaved and currently available are based on the SOSIP framework (40, 41). Most recombinant Env constructs are uncleaved and many use a heterologous trimerization motif to impose trimer symmetry (42, 43). The b12H mice should be useful to assess vaccine candidates that selectively recruit B cells recognizing the most relevant epitope.
A future challenge will be to learn how to train germline B cells to adopt specificity to achieve both high affinity and the ability to neutralize HIV. In this effort, mice carrying germline versions of b12 HL and other bNAbs will be of special value. These B cells, though, lack detectable affinity for HIV (21, 44). Recent progress on rational immunogen design has suggested a pathway to recruit such cells (45). However, our study of b12H mice suggests that we still have much to learn from this model about how to extend moderate binding energy of BCRs to promote production of Ab able to neutralize HIV.
The authors have no financial conflicts of interest.
We thank the The Scripps Research Institute Mouse Genetics Facility, Patrick Skog (The Scripps Research Institute) for excellent technical assistance, and Roberta Pelanda (National Jewish Health) for the pVKR2neo targeting vector.
This work was supported by National Institutes of Health Research Grants R01AI073148 and UO1AI078224 and by the International AIDS Vaccine Initiative Neutralizing Antibody Center and the Ragon Institute.
Abbreviations used in this article:
- bone marrow
- broadly neutralizing Ab
- embryonic stem
- lymph node
- marginal zone
- Received May 14, 2013.
- Accepted July 12, 2013.
- Copyright © 2013 by The American Association of Immunologists, Inc.