Gene Editing of Primary Rhesus Macaque B Cells.

B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy accessibility via peripheral blood, and amenability to simple adoptive transfers have made them an attractive target for gene editing approaches to express recombinant antibodies or other therapeutic proteins. The gene editing of mouse and human primary B cells is efficient, and mouse models for in vivo studies have shown promise, but feasibility and scalability for larger animal models have so far not been demonstrated. We, therefore, developed a protocol to edit rhesus macaque primary B cells in vitro to enable such studies. We report conditions for in vitro culture and gene-editing of primary rhesus macaque B cells from peripheral blood mononuclear cells or splenocytes using CRISPR/Cas9. To achieve the targeted integration of large (<4.5 kb) cassettes, a fast and efficient protocol was included for preparing recombinant adeno-associated virus serotype 6 as a homology-directed repair template using a tetracycline-enabled self-silencing adenoviral helper vector. These protocols enable the study of prospective B cell therapeutics in rhesus macaques.

Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer.

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterize Ab1303 and Ab1573, heterologously-neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding is observed only when Env trimers are not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures show that both antibodies recognize the CD4bs on Env trimer with an 'occluded-open' conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation includes outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, does not exhibit V1V2 displacement, 4-stranded gp120 bridging sheet, or co-receptor binding site exposure. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggest an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

Sequential immunization of macaques elicits heterologous neutralizing antibodies targeting the V3-glycan patch of HIV-1 Env.

Broadly neutralizing antibodies (bNAbs) against HIV-1 develop after prolonged virus and antibody coevolution. Previous studies showed that sequential immunization with a V3-glycan patch germline-targeting HIV-1 envelope trimer (Env) followed by variant Envs can reproduce this process in mice carrying V3-glycan bNAb precursor B cells. However, eliciting bNAbs in animals with polyclonal antibody repertoires is more difficult. We used a V3-glycan immunogen multimerized on virus-like particles (VLPs), followed by boosting with increasingly native-like Env-VLPs, to elicit heterologous neutralizing antibodies in nonhuman primates (NHPs). Structures of antibody/Env complexes after prime and boost vaccinations demonstrated target epitope recognition with apparent maturation to accommodate glycans. However, we also observed increasing off-target antibodies with boosting. Eight vaccinated NHPs were subsequently challenged with simian-human immunodeficiency virus (SHIV), and seven of eight animals became infected. The single NHP that remained uninfected after viral challenge exhibited one of the lowest neutralization titers against the challenge virus. These results demonstrate that more potent heterologous neutralization resulting from sequential immunization is necessary for protection in this animal model. Thus, improved prime-boost regimens to increase bNAb potency and stimulate other immune protection mechanisms are essential for developing anti­HIV-1 vaccines.

Antibody elicited by HIV-1 immunogen vaccination in macaques displaces Env fusion peptide and destroys a neutralizing epitope.

HIV-1 vaccine design aims to develop an immunogen that elicits broadly neutralizing antibodies against a desired epitope, while eliminating responses to off-target regions of HIV-1 Env. We report characterization of Ab1245, an off-target antibody against the Env gp120-gp41 interface, from V3-glycan patch immunogen-primed and boosted macaques. A 3.7 Å cryo-EM structure of an Ab1245-Env complex reveals one Ab1245 Fab binding asymmetrically to Env trimer at the gp120-gp41 interface using its long CDRH3 to mimic regions of gp41. The mimicry includes positioning of a CDRH3 methionine into the gp41 tryptophan clasp, resulting in displacement of the fusion peptide and fusion peptide-proximal region. Despite fusion peptide displacement, Ab1245 is non-neutralizing even at high concentrations, raising the possibility that only two fusion peptides per trimer are required for viral-host membrane fusion. These structural analyses facilitate immunogen design to prevent elicitation of Ab1245-like antibodies that block neutralizing antibodies against the fusion peptide.

Sequencing, cloning, and antigen binding analysis of monoclonal antibodies isolated from single mouse B cells.

The analysis of B cell receptors (BCR) from single B cells is crucial to understanding humoral immune responses. Here, we describe a protocol for the sequencing, cloning, and characterization of antibody genes that encode BCRs. We used this method to analyze the BCRs of different mouse B cell populations for somatic hypermutations, clonal and phylogenic relationships, and their affinity for cognate antigen. For complete details on the use and execution of this protocol, please refer to Viant et al. (2020).

Antibody Affinity Shapes the Choice between Memory and Germinal Center B Cell Fates.

Immunological memory is required for protection against repeated infections and is the basis of all effective vaccines. Antibodies produced by memory B cells play an essential role in many of these responses. We have combined lineage tracing with antibody cloning from single B cells to examine the role of affinity in B cell selection into germinal centers (GCs) and the memory B cell compartment in mice immunized with an HIV-1 antigen. We find that contemporaneously developing memory and GC B cells differ in their affinity for antigen throughout the immune response. Whereas GC cells and their precursors are enriched in antigen binding, memory B cells are not. Thus, the polyclonal memory B cell compartment is composed of B cells that were activated during the immune response but whose antigen binding affinity failed to support further clonal expansion in the GC.

A broadly neutralizing macaque monoclonal antibody against the HIV-1 V3-Glycan patch.

A small fraction of HIV-1- infected humans develop broadly neutralizing antibodies (bNAbs) against HIV-1 that protect macaques from simian immunodeficiency HIV chimeric virus (SHIV). Similarly, a small number of macaques infected with SHIVs develop broadly neutralizing serologic activity, but less is known about the nature of simian antibodies. Here, we report on a monoclonal antibody, Ab1485, isolated from a macaque infected with SHIVAD8 that developed broadly neutralizing serologic activity targeting the V3-glycan region of HIV-1 Env. Ab1485 neutralizes 38.1% of HIV-1 isolates in a 42-pseudovirus panel with a geometric mean IC50 of 0.055 µg/mLl and SHIVAD8 with an IC50 of 0.028 µg/mLl. Ab1485 binds the V3-glycan epitope in a glycan-dependent manner. A 3.5 Å cryo-electron microscopy structure of Ab1485 in complex with a native-like SOSIP Env trimer showed conserved contacts with the N332gp120 glycan and gp120 GDIR peptide motif, but in a distinct Env-binding orientation relative to human V3/N332gp120 glycan-targeting bNAbs. Intravenous infusion of Ab1485 protected macaques from a high dose challenge with SHIVAD8. We conclude that macaques can develop bNAbs against the V3-glycan patch that resemble human V3-glycan bNAbs.

Isolation of single HIV-1 Envelope specific B cells and antibody cloning from immunized rhesus macaques.

Antibody cloning from single B cells is an essential tool for characterizing humoral immune responses and obtaining valuable therapeutic and analytical reagents. Antibody cloning from individuals with high serologic titers to HIV-1, Influenza, Malaria and ZIKV has led to new insights that inform vaccine design efforts. In contrast to humans and mice, less is known about antibody cloning from single B cells in macaques. Here, we describe a protocol to identify and purify single antigen-specific macaque B cells, and subsequently clone and produce macaque monoclonal antibodies. The sorting strategy requires the use of a combination of fluorochrome labeled antigens and omission of anti-IgG antibodies that can interfere with antigen binding and vice versa. Optimized methods for macaque antibody gene amplification, DNA preparation for antibody production and antibody screening by ELISA are also presented.

Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques.

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires.

Comparative Analysis between the In Vivo Biodistribution and Therapeutic Efficacy of Adipose-Derived Mesenchymal Stromal Cells Administered Intraperitoneally in Experimental Colitis.

Mesenchymal stem cells (MSCs) have emerged as a promising treatment for inflammatory diseases. The immunomodulatory effect of MSCs takes place both by direct cell-to-cell contact and by means of soluble factors that leads to an increased accumulation of regulatory immune cells at the sites of inflammation. Similar efficacy of MSCs has been described regardless of the route of administration used, the inflammation conditions and the major histocompatibility complex context. These observations raise the question of whether the migration of the MSCs to the inflamed tissues is a pre-requisite to achieve their beneficial effect. To address this, we examined the biodistribution and the efficacy of intraperitoneal luciferase-expressing human expanded adipose-derived stem cells (Luci-eASCs) in a mouse model of colitis. Luci-eASC-infused mice were stratified according to their response to the Luci-eASC treatment. According to the stratification criteria, there was a tendency to increase the bioluminescence signal in the intestine at the expense of a decrease in the bioluminescence signal in the liver in the "responder" mice. These data thus suggest that the accumulation of the eASCs to the inflamed tissues is beneficial for achieving an optimal modulation of inflammation.

Structural characterization of a highly-potent V3-glycan broadly neutralizing antibody bound to natively-glycosylated HIV-1 envelope.

Broadly neutralizing antibodies (bNAbs) isolated from HIV-1-infected individuals inform HIV-1 vaccine design efforts. Developing bNAbs with increased efficacy requires understanding how antibodies interact with the native oligomannose and complex-type N-glycan shield that hides most protein epitopes on HIV-1 envelope (Env). Here we present crystal structures, including a 3.8-Å X-ray free electron laser dataset, of natively glycosylated Env trimers complexed with BG18, the most potent V3/N332gp120 glycan-targeting bNAb reported to date. Our structures show conserved contacts mediated by common D gene-encoded residues with the N332gp120 glycan and the gp120 GDIR peptide motif, but a distinct Env-binding orientation relative to PGT121/10-1074 bNAbs. BG18's binding orientation provides additional contacts with N392gp120 and N386gp120 glycans near the V3-loop base and engages protein components of the V1-loop. The BG18-natively-glycosylated Env structures facilitate understanding of bNAb-glycan interactions critical for using V3/N332gp120 bNAbs therapeutically and targeting their epitope for immunogen design.

The microanatomic segregation of selection by apoptosis in the germinal center.

B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC, with up to half of all GC B cells dying every 6 hours. Moreover, programmed cell death is differentially regulated in the light zone and the dark zone: Light-zone B cells die by default if they are not positively selected, whereas dark-zone cells die when their antigen receptors are damaged by activation-induced cytidine deaminase.

Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo.

Induction of broadly neutralizing antibodies (bNAbs) by HIV-1 envelope glycoprotein immunogens would be a major advance toward an effective vaccine. A critical step in this process is the activation of naive B cells expressing germline (gl) antibody precursors that have the potential to evolve into bNAbs. Here, we reengineered the BG505 SOSIP.664 glycoprotein to engage gl precursors of bNAbs that target either the trimer apex or the CD4-binding site. The resulting BG505 SOSIP.v4.1-GT1 trimer binds multiple bNAb gl precursors in vitro. Immunization experiments in knock-in mice expressing gl-VRC01 or gl-PGT121 show that this trimer activates B cells in vivo, resulting in the secretion of specific antibodies into the sera. A crystal structure of the gl-targeting trimer at 3.2-Å resolution in complex with neutralizing antibodies 35O22 and 9H+109L reveals a native-like conformation and the successful incorporation of design features associated with binding of multiple gl-bNAb precursors.

Biodistribution and Efficacy of Human Adipose-Derived Mesenchymal Stem Cells Following Intranodal Administration in Experimental Colitis.

Mesenchymal stem cells (MSCs) have a large potential in cell therapy for treatment of inflammatory and autoimmune diseases, thanks to their immunomodulatory properties. The encouraging results in animal models have initiated the translation of MSC therapy to clinical trials. In cell therapy protocols with MSCs, administered intravenously, several studies have shown that a small proportion of infused MSCs can traffic to the draining lymph nodes (LNs). This is accompanied with an increase of different types of regulatory immune cells in the LNs, suggesting the importance of migration of MSCs to the LNs in order to contribute to immunomodulatory response. Intranodal (IN), also referred as intralymphatic, injection of cells, like dendritic cells, is being proposed in the clinic for the treatment of cancer and allergy, showing that this route of administration is clinically safe and efficient. In this study, we investigated, for the first time, the biodistribution and the efficacy of Luciferase+ adipose-derived MSCs (Luci-eASCs), infused through the inguinal LNs (iLNs), in normal mice and in inflamed mice with colitis. Most of the Luci-eASCs remain in the iLNs and in the adipose tissue surrounding the inguinal LNs. A small proportion of Luci-eASCs can migrate to other locations within the lymphatic system and to other tissues and organs, having a preferential migration toward the intestine in colitic mice. Our results show that the infused Luci-eASCs protected 58% of the mice against induced colitis. Importantly, a correlation between the response to eASC treatment and a higher accumulation of eASCs in popliteal, parathymic, parathyroid, and mesenteric LNs were found. Altogether, these results suggest that IN administration of eASCs is feasible and may represent an effective strategy for cell therapy protocols with human adipose-derived MSCs in the clinic for the treatment of immune-mediated disorders.

Progress toward active or passive HIV-1 vaccination.

AIDS is a preventable disease. Nevertheless, according to UNAIDS, 2.1 million individuals were infected with HIV-1 in 2015 worldwide. An effective vaccine is highly desirable. Most vaccines in clinical use today prevent infection because they elicit antibodies that block pathogen entry. Consistent with this general rule, studies in experimental animals have shown that broadly neutralizing antibodies to HIV-1 can prevent infection, suggesting that a vaccine that elicits such antibodies would be protective. However, despite significant efforts over the last 30 years, attempts to elicit broadly HIV-1 neutralizing antibodies by vaccination failed until recent experiments in genetically engineered mice were finally successful. Here, we review the key breakthroughs and remaining obstacles to the development of active and passive HIV-1 vaccines.

HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies.

Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.

Sequential Immunization Elicits Broadly Neutralizing Anti-HIV-1 Antibodies in Ig Knockin Mice.

A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.

C/EBPß and Nuclear Factor of Activated T Cells Differentially Regulate Adamts-1 Induction by Stimuli Associated with Vascular Remodeling.

Emerging evidence indicates that the metalloproteinase Adamts-1 plays a significant role in the pathophysiology of vessel remodeling, but little is known about the signaling pathways that control Adamts-1 expression. We show that vascular endothelial growth factor (VEGF), angiotensin-II, interleukin-1ß, and tumor necrosis factor α, stimuli implicated in pathological vascular remodeling, increase Adamts-1 expression in endothelial and vascular smooth muscle cells. Analysis of the intracellular signaling pathways implicated in this process revealed that VEGF and angiotensin-II upregulate Adamts-1 expression via activation of differential signaling pathways that ultimately promote functional binding of the NFAT or C/EBPß transcription factors, respectively, to the Adamts-1 promoter. Infusion of mice with angiotensin-II triggered phosphorylation and nuclear translocation of C/EBPß proteins in aortic cells concomitantly with an increase in the expression of Adamts-1, further underscoring the importance of C/EBPß signaling in angiotensin-II-induced upregulation of Adamts-1. Similarly, VEGF promoted NFAT activation and subsequent Adamts-1 induction in aortic wall in a calcineurin-dependent manner. Our results demonstrate that Adamts-1 upregulation by inducers of pathological vascular remodeling is mediated by specific signal transduction pathways involving NFAT or C/EBPß transcription factors. Targeting of these pathways may prove useful in the treatment of vascular disease.

Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knockin Mice.

A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.

Retinoid X receptors orchestrate osteoclast differentiation and postnatal bone remodeling.

Osteoclasts are bone-resorbing cells that are important for maintenance of bone remodeling and mineral homeostasis. Regulation of osteoclast differentiation and activity is important for the pathogenesis and treatment of diseases associated with bone loss. Here, we demonstrate that retinoid X receptors (RXRs) are key elements of the transcriptional program of differentiating osteoclasts. Loss of RXR function in hematopoietic cells resulted in formation of giant, nonresorbing osteoclasts and increased bone mass in male mice and protected female mice from bone loss following ovariectomy, which induces osteoporosis in WT females. The increase in bone mass associated with RXR deficiency was due to lack of expression of the RXR-dependent transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB) in osteoclast progenitors. Evaluation of osteoclast progenitor cells revealed that RXR homodimers directly target and bind to the Mafb promoter, and this interaction is required for proper osteoclast proliferation, differentiation, and activity. Pharmacological activation of RXRs inhibited osteoclast differentiation due to the formation of RXR/liver X receptor (LXR) heterodimers, which induced expression of sterol regulatory element binding protein-1c (SREBP-1c), resulting in indirect MAFB upregulation. Our study reveals that RXR signaling mediates bone homeostasis and suggests that RXRs have potential as targets for the treatment of bone pathologies such as osteoporosis.