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The immunoglobulin locus of B cells can be reprogrammed by genome editing to produce custom or non-natural antibodies that are not induced by immunization. However, current strategies for antibody reprogramming require complex expression cassettes and do not allow for customization of the constant region of the antibody. Here we show that human B cells can be edited at the immunoglobulin heavy-chain locus to express heavy-chain-only antibodies that support alterations to both the fragment crystallizable domain and the antigen-binding domain, which can be based on both antibody and non-antibody components. Using the envelope protein (Env) from the human immunodeficiency virus as a model antigen, we show that B cells edited to express heavy-chain antibodies to Env support the regulated expression of B cell receptors and antibodies through alternative splicing and that the cells respond to the Env antigen in a tonsil organoid model of immunization. This strategy allows for the reprogramming of human B cells to retain the potential for in vivo amplification while producing molecules with flexibility of composition beyond that of standard antibodies.
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When a laminate or transparent coating is applied to the surface of a colored slab, there may be a significant change in the color. A reason for the change in color is internal reflection at the slab surface and thus greater selective absorption. The current work develops a random walk model to calculate the reflectance factor of a slab that includes internal reflection at the slab surface to predict the change in color of the bulk reflectance with application of a laminate. The CIELAB colors are calculated from the reflectance, and the model shows that there is not much change in hue but there is significant change in the saturation and lightness: the saturation increases and the lightness decreases. A comparison of the reflectance factor as predicted by the model is compared to a Monte Carlo simulation and shown to have good agreement.
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With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor-binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors could be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the Spike receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped vesicular stomatitis virus vectors and replication-competent viruses. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed in the Spike proteins from Beta, Gamma, and Omicron (BA.1 and BA.5). The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. IMPORTANCE The increasing resistance of SARS-CoV-2 variants to therapeutic antibodies has highlighted the need for new treatment options, especially in individuals who do not respond to vaccination. Receptor decoys that block viral entry are an attractive approach because of the presumed high bar to developing viral resistance. Here, we compare two entry inhibitors based on derivatives of the ACE2 receptor, or engineered peptides that bind to the receptor-binding pocket of the SARS-CoV-2 Spike protein. In each case, the inhibitors were fused to immunoglobulin Fc domains, which can further enhance therapeutic properties, and compared for activity against different SARS-CoV-2 variants. Potent inhibition against multiple SARS-CoV-2 variants was demonstrated in vitro, and even relatively low single doses of optimized reagents provided some protection in a mouse model, confirming their potential as an alternative to antibody therapies.
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COVID-19 , Inhibidores de Fusión de VIH , Animales , Ratones , Humanos , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Ratones Transgénicos , Péptidos/farmacologíaRESUMEN
We describe a genome editing strategy to reprogram the immunoglobulin heavy chain (IgH) locus of human B cells to express custom molecules that respond to immunization. These heavy chain antibodies (HCAbs) comprise a custom antigen-recognition domain linked to an Fc domain derived from the IgH locus and can be differentially spliced to express either B cell receptor (BCR) or secreted antibody isoforms. The HCAb editing platform is highly flexible, supporting antigen-binding domains based on both antibody and non-antibody components, and also allowing alterations in the Fc domain. Using HIV Env protein as a model antigen, we show that B cells edited to express anti-Env HCAbs support the regulated expression of both BCRs and antibodies, and respond to Env antigen in a tonsil organoid model of immunization. In this way, human B cells can be reprogrammed to produce customized therapeutic molecules with the potential for in vivo amplification.
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We describe a genome editing strategy to reprogram the immunoglobulin heavy chain (IgH) locus of human B cells to express custom molecules that respond to immunization. These heavy chain antibodies (HCAbs) comprise a custom antigen-recognition domain linked to an Fc domain derived from the IgH locus and can be differentially spliced to express either B cell receptor (BCR) or secreted antibody isoforms. The HCAb editing platform is highly flexible, supporting antigen-binding domains based on both antibody and non-antibody components, and also allowing alterations in the Fc domain. Using HIV Env protein as a model antigen, we show that B cells edited to express anti-Env HCAbs support the regulated expression of both BCRs and antibodies, and respond to Env antigen in a tonsil organoid model of immunization. In this way, human B cells can be reprogrammed to produce customized therapeutic molecules with the potential for in vivo amplification.
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Adeno-associated virus serotype 6 (AAV6) is a valuable reagent for genome editing of hematopoietic cells due to its ability to serve as a homology donor template. However, a comprehensive study of AAV6 transduction of hematopoietic cells in culture, with the goal of maximizing ex vivo genome editing, has not been reported. Here, we evaluated how the presence of serum, culture volume, transduction time, and electroporation parameters could influence AAV6 transduction. Based on these results, we identified an optimized protocol for genome editing of human lymphocytes based on a short, highly concentrated AAV6 transduction in the absence of serum, followed by electroporation with a targeted nuclease. In human CD4+ T cells and B cells, this protocol improved editing rates up to 7-fold and 21-fold, respectively, when compared to standard AAV6 transduction protocols described in the literature. As a result, editing frequencies could be maintained using 50- to 100-fold less AAV6, which also reduced cellular toxicity. Our results highlight the important contribution of cell culture conditions for ex vivo genome editing with AAV6 vectors and provide a blueprint for improving AAV6-mediated homology-directed editing of human T and B cells.
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Cell therapies based on reprogrammed adaptive immune cells have great potential as "living drugs." As first demonstrated clinically for engineered chimeric antigen receptor (CAR) T cells, the ability of such cells to undergo clonal expansion in response to an antigen promotes both self-renewal and self-regulation in vivo. B cells also have the potential to be developed as immune cell therapies, but engineering their specificity and functionality is more challenging than for T cells. In part, this is due to the complexity of the immunoglobulin (Ig) locus, as well as the requirement for regulated expression of both cell surface B cell receptor and secreted antibody isoforms, in order to fully recapitulate the features of natural antibody production. Recent advances in genome editing are now allowing reprogramming of B cells by site-specific engineering of the Ig locus with preformed antibodies. In this review, we discuss the potential of engineered B cells as a cell therapy, the challenges involved in editing the Ig locus and the advances that are making this possible, and envision future directions for this emerging field of immune cell engineering.
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Linfocitos B/metabolismo , Sistemas CRISPR-Cas , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Edición Génica , Terapia Genética/métodos , Inmunoterapia/métodos , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Linfocitos B/inmunología , Ingeniería Celular , Reprogramación Celular/genética , Reprogramación Celular/inmunología , Regulación de la Expresión Génica , Ingeniería Genética , Humanos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein-pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV), and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein's function. Here, we optimized concentration methods for SARS-CoV-2 Spike-pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers but had no impact on sensitivity to convalescent serum. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions. IMPORTANCE Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant arising early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration if higher-titer vectors are required.
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Vectores Genéticos/fisiología , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Anticuerpos Neutralizantes/inmunología , Línea Celular , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Lentivirus/genética , Mutación , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Virus de la Estomatitis Vesicular Indiana/genética , Carga Viral/genéticaRESUMEN
A transmission point spread function characterizes the spatial distribution of light transmitted through a turbid slab. Because of scatter due to turbidity, light diffuses and the distribution of photons on exiting the slab may be very different from that upon entering the slab. Using the multiple-path model of reflection and transmission, the transmission point spread function and related modulation transfer function are derived. The analytic expression for the point spread function is compared to a Monte Carlo simulation of photon transmission. The transmission modulation transfer function is used to define a measure of visibility through the turbid slab-a contrast transfer function.
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Adeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem and progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 and AAVS1 loci in human HSPCs and cell lines. As a control, we used AAV6, which effectively edits HSPCs but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc-finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results, therefore, do not support that clade F AAVs can perform high-efficiency genome editing in the absence of a DSB.
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Roturas del ADN de Doble Cadena , Dependovirus/fisiología , Edición Génica/métodos , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/citología , Células Cultivadas , Dependovirus/clasificación , Dependovirus/genética , Marcación de Gen , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Células HeLa , Células Madre Hematopoyéticas/metabolismo , Recombinación Homóloga , Humanos , Células K562 , Receptores CCR5/genética , Ensamble de VirusRESUMEN
The diffusion of light within paper has an effect on the colors in a halftone image and must be taken into consideration in modeling halftone color. The diffusion can be accounted for by using the point spread function or equivalently the modulation transfer function (MTF) of the paper. A novel method of measurement of the paper's MTF, called the bar-target series expansion method, is proposed here. The experimental results of the method confirm that the shape of the MTF is an exponential function, which corresponds to a Lorentzian line spread function as predicted by the multiple-path model of reflection.
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About 10% of Americans over the age of 60 experience elder abuse (EA), but EA is frequently under detected. As the primary responders for alleged EA cases, Adult Protective Service (APS) caseworkers are ideally positioned to improve detection and intervention. The Opening the Door (OTD) training was developed in a multidisciplinary setting and designed in collaboration with APS workers to enhance their engagement skills in potential EA cases and thus maximize the possibility of disclosure. Rooted in a relational approach and utilizing adult learning theory, it is divided into three main topics: Preparing for Engagement, Engagement Practice, and Processing Engagement. The eight-hour training was piloted with three groups of urban APS caseworkers with a wide range of work experience. Pre- and post-tests and case reflections indicate a positive impact on caseworkers' sense of self-efficacy, while focus groups indicate the necessity for ongoing provision of trainings and development of additional supports for APS caseworkers. Future work could further explore the efficacy of the training and expand its reach.
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Abuso de Ancianos/diagnóstico , Abuso de Ancianos/prevención & control , Autoeficacia , Trabajadores Sociales/educación , Adulto , Anciano , Educación Profesional/métodos , Femenino , Grupos Focales/métodos , Agencias Gubernamentales , Humanos , Masculino , Ciudad de Nueva York , Proyectos PilotoRESUMEN
We present the Interview for Decisional Abilities (IDA), a semi-structured tool for use by adult protective services (APS) workers as part of their comprehensive assessments of clients. The IDA was created in response to a Federal mandate to standardize the procedures and competencies of APS agencies with a view to improving client assessments and facilitating cross-jurisdictional research on adult mistreatment. The proximal aim of the IDA is to guide workers in gathering information on the ability of suspected victims of adult mistreatment to make decisions about the risks they face.
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Abuso de Ancianos/diagnóstico , Entrevista Psicológica , Anciano , Agencias Gubernamentales , Humanos , Trabajadores SocialesRESUMEN
We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.
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Reacciones Antígeno-Anticuerpo/genética , Linfocitos B/inmunología , Edición Génica/métodos , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Sistemas CRISPR-Cas , Línea Celular , Citidina Desaminasa/metabolismo , Anticuerpos Anti-VIH/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
The Yule-Nielsen effect is an influence on halftone color caused by the diffusion of light within the paper upon which the halftone ink is printed. The diffusion can be characterized by a point spread function. In this paper, a point spread function for paper is derived using the multiple-path model of reflection. This model treats the interaction of light with turbid media as a random walk. Using the multiple-path point spread function, a general expression is derived for the average reflectance of light from a frequency-modulated halftone, in which dot size is constant and the number of dots is varied, with the arrangement of dots random. It is also shown that the line spread function derived from the multiple-path model has the form of a Lorentzian function.
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Advances in gene therapy technologies, particularly in gene editing, are suggesting new avenues for the treatment of human immunodeficiency virus and other infectious diseases. This article outlines recent developments in antiviral gene therapies, including those based on the disruption of entry receptors or that target viral genomes using targeted nucleases, such as the CRISPR/Cas9 system. In addition, new ways to express circulating antiviral factors, such as antibodies, and approaches to harness and engineer the immune system to provide an antiviral effect that is not naturally achieved are described.
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Enfermedades Transmisibles/terapia , Terapia Genética , Infecciones por VIH/terapia , Infecciones por VIH/virología , VIH/genética , Sistemas CRISPR-Cas , Resistencia a la Enfermedad/genética , Edición Génica , Expresión Génica , Marcación de Gen , Ingeniería Genética , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos/genética , Genoma Viral , Humanos , Inmunomodulación/genética , Receptores del VIH/genética , Receptores del VIH/metabolismo , TransgenesRESUMEN
Experimental results are presented of the spectral reflectance of a dyed fabric as analyzed by a multiple-path model of reflection. The multiple-path model provides simple analytic expressions for reflection and transmission of turbid media by applying the Beer-Lambert law to each path through the medium and summing over all paths, each path weighted by its probability. The path-length probability is determined by a random-walk analysis. The experimental results presented here show excellent agreement with predictions made by the model.
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Adeno-associated virus (AAV) is a replication-deficient parvovirus that is extensively used as a gene therapy vector. CD8+ T-cell responses against the AAV capsid protein can, however, affect therapeutic efficacy. Little is known about the in vivo mechanism that leads to the crosspriming of CD8+ T cells against the input viral capsid antigen. In this study, we report that the Toll-like receptor 9 (TLR9)-MyD88 pattern-recognition receptor pathway is uniquely capable of initiating this response. By contrast, the absence of TLR2, STING, or the addition of TLR4 agonist has no effect. Surprisingly, both conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) are required for the crosspriming of capsid-specific CD8+ T cells, whereas other antigen-presenting cells are not involved. TLR9 signaling is specifically essential in pDCs but not in cDCs, indicating that sensing of the viral genome by pDCs activates cDCs in trans to cross-present capsid antigen during CD8+ T-cell activation. Cross-presentation and crosspriming depend not only on TLR9, but also on interferon type I signaling, and both mechanisms can be inhibited by administering specific molecules to prevent induction of capsid-specific CD8+ T cells. Thus, these outcomes directly point to therapeutic interventions and demonstrate that innate immune blockade can eliminate unwanted immune responses in gene therapy.
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Linfocitos T CD8-positivos/inmunología , Proteínas de la Cápside/inmunología , Células Dendríticas/inmunología , Dependovirus/inmunología , Activación de Linfocitos , Células Plasmáticas/inmunología , Animales , Proteínas de la Cápside/genética , Dependovirus/genética , Terapia Genética , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunologíaRESUMEN
Development of antibodies (inhibitors) against coagulation factor VIII (FVIII) is a major complication of intravenous replacement therapy in haemophilia A (HA). Current immune tolerance induction (ITI) regimens are not universally effective. Rituximab, a B cell-depleting antibody against CD20, has shown mixed results for inhibitor reversal in patients. This study aims to develop a combinatorial therapy for inhibitor reversal in HA, using anti-murine CD20 (anti-mCD20) antibody and rapamycin, which targets both B and T cell responses. Additionally, it extensively characterises the role of the IgG backbone in B cell depletion by anti-CD20 antibodies. For this, inhibitors were generated in BALB/c-HA mice by weekly IV injection of FVIII. Subsequently, anti-mCD20 (18B12) with IgG2a or IgG1 backbone was injected IV in two doses three weeks apart and B cell depletion and recovery was characterised. Rapamycin was administered orally 3x/week (for 1 month) while continuing FVIII injections. Altering the IgG backbone of anti-mCD20 from IgG2a to IgG1 reduced overall depletion of B cells (including memory B cells), and marginal zone, B-10, and B-1b cells were specifically unaffected. While neither antibody was effective alone, in combination with rapamycin, anti-mCD20 IgG2a but not IgG1 was able to reverse inhibitors in HA mice. This regimen was particularly effective for starting titres of ~10 BU. Although IgG1 anti-mCD20 spared potentially tolerogenic B cell subsets, IgG2a directed sustained hyporesponsiveness when administered in conjunction with rapamycin. This regimen represents a promising treatment for inhibitor reversal in HA, as both of these compounds have been extensively used in human patients.