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1.
Nat Commun ; 14(1): 7897, 2023 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-38036525

RESUMEN

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employ computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant receptor binding domain. These engineered proteins bind with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interact with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicit sera with broad betacoronavirus reactivity and protect as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.


Asunto(s)
Anticuerpos Antivirales , SARS-CoV-2 , Humanos , Animales , Ratones , Epítopos , Epítopos Inmunodominantes , Péptidos , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Neutralizantes
2.
PLoS Pathog ; 19(5): e1011401, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37196027

RESUMEN

A major goal for the development of vaccines against rapidly mutating viruses, such as influenza or HIV, is to elicit antibodies with broad neutralization capacity. However, B cell precursors capable of maturing into broadly neutralizing antibodies (bnAbs) can be rare in the immune repertoire. Due to the stochastic nature of B cell receptor (BCR) rearrangement, a limited number of third heavy chain complementary determining region (CDRH3) sequences are identical between different individuals. Thus, in order to successfully engage broadly neutralizing antibody precursors that rely on their CDRH3 loop for antigen recognition, immunogens must be able to tolerate sequence diversity in the B cell receptor repertoire across an entire vaccinated population. Here, we present a combined experimental and computational approach to identify BCRs in the human repertoire with CDRH3 loops predicted to be engaged by a target immunogen. For a given antibody/antigen pair, deep mutational scanning was first used to measure the effect of CDRH3 loop substitution on binding. BCR sequences, isolated experimentally or generated in silico, were subsequently evaluated to identify CDRH3 loops expected to be bound by the candidate immunogen. We applied this method to characterize two HIV-1 germline-targeting immunogens and found differences in the frequencies with which they are expected to engage target B cells, thus illustrating how this approach can be used to evaluate candidate immunogens towards B cell precursors engagement and to inform immunogen optimization strategies for more effective vaccine design.


Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Humanos , Anticuerpos Anti-VIH , Anticuerpos Neutralizantes , Linfocitos B , Anticuerpos ampliamente neutralizantes , Receptores de Antígenos de Linfocitos B/genética
3.
bioRxiv ; 2023 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-36909627

RESUMEN

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employed computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant RBD. These engineered proteins bound with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interacted with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicited sera with broad betacoronavirus reactivity and protected as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.

4.
Commun Biol ; 5(1): 271, 2022 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-35347236

RESUMEN

The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.


Asunto(s)
Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase I , Animales , Antígenos HLA , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoglobulinas/metabolismo , Células Asesinas Naturales , Ratones , Péptidos/metabolismo , Señales de Clasificación de Proteína , Antígenos HLA-E
5.
Cell Rep ; 36(7): 109561, 2021 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-34407396

RESUMEN

Elicitation of broadly neutralizing antibodies (bnAbs) by an HIV vaccine will involve priming the immune system to activate antibody precursors, followed by boosting immunizations to select for antibodies with functional features required for neutralization breadth. The higher the number of acquired mutations necessary for function, the more convoluted are the antibody developmental pathways. HIV bnAbs acquire a large number of somatic mutations, but not all mutations are functionally important. In this study, we identify a minimal subset of mutations sufficient for the function of the naturally occurring V3-glycan bnAb DH270.6. Using antibody library screening, candidate envelope immunogens that interact with DH270.6-like antibodies containing this set of key mutations are identified and selected in vitro. Our results demonstrate that less complex B cell evolutionary pathways than those naturally observed exist for the induction of HIV bnAbs by vaccination, and they establish rational approaches to identify boosting candidate immunogens.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Linfocitos B/citología , Linaje de la Célula , Mutación/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Línea Celular , Humanos , Mutagénesis/genética , Unión Proteica , Vacunación , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química
6.
Nat Chem Biol ; 16(8): 826-833, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32424303

RESUMEN

Here we generate fluorescence resonance energy transfer biosensors for guanine exchange factors (GEFs) by inserting a fluorescent protein pair in a structural 'hinge' common to many GEFs. Fluorescent biosensors can map the activation of signaling molecules in space and time, but it has not been possible to quantify how different activation events affect one another or contribute to a specific cell behavior. By imaging the GEF biosensors in the same cells as red-shifted biosensors of Rho GTPases, we can apply partial correlation analysis to parse out the extent to which each GEF contributes to the activation of a specific GTPase in regulating cell movement. Through analysis of spontaneous cell protrusion events, we identify when and where the GEF Asef regulates the GTPases Cdc42 and Rac1 to control cell edge dynamics. This approach exemplifies a powerful means to elucidate the real-time connectivity of signal transduction networks.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Factores de Intercambio de Guanina Nucleótido/metabolismo , Secuencia de Aminoácidos/genética , Técnicas Biosensibles/métodos , Unión Proteica/genética , Homología de Secuencia de Aminoácido , Transducción de Señal/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismo
7.
J Cell Biol ; 218(9): 3077-3097, 2019 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-31420453

RESUMEN

Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0-2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1.


Asunto(s)
Microtúbulos/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Proteína de Unión al GTP rhoA/metabolismo , Familia-src Quinasas/metabolismo , Animales , Técnicas Biosensibles , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Microtúbulos/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteína de Unión al GTP rhoA/genética , Familia-src Quinasas/genética
8.
J Cell Biol ; 218(9): 3153-3160, 2019 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-31444239

RESUMEN

Lattice light-sheet microscopy (LLSM) is valuable for its combination of reduced photobleaching and outstanding spatiotemporal resolution in 3D. Using LLSM to image biosensors in living cells could provide unprecedented visualization of rapid, localized changes in protein conformation or posttranslational modification. However, computational manipulations required for biosensor imaging with LLSM are challenging for many software packages. The calculations require processing large amounts of data even for simple changes such as reorientation of cell renderings or testing the effects of user-selectable settings, and lattice imaging poses unique challenges in thresholding and ratio imaging. We describe here a new software package, named ImageTank, that is specifically designed for practical imaging of biosensors using LLSM. To demonstrate its capabilities, we use a new biosensor to study the rapid 3D dynamics of the small GTPase Rap1 in vesicles and cell protrusions.


Asunto(s)
Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Procesamiento de Imagen Asistido por Computador , Transducción de Señal , Programas Informáticos , Proteínas de Unión a Telómeros/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Microscopía Fluorescente , Complejo Shelterina , Proteínas de Unión a Telómeros/genética
9.
J Mol Biol ; 415(1): 175-92, 2012 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-22061265

RESUMEN

Computational grafting of functional motifs onto scaffold proteins is a promising way to engineer novel proteins with pre-specified functionalities. Typically, protein grafting involves the transplantation of protein side chains from a functional motif onto structurally homologous regions of scaffold proteins. Using this approach, we previously transplanted the human immunodeficiency virus 2F5 and 4E10 epitopes onto heterologous proteins to design novel "epitope-scaffold" antigens. However, side-chain grafting is limited by the availability of scaffolds with compatible backbone for a given epitope structure and offers no route to modify backbone structure to improve mimicry or binding affinity. To address this, we report here a new and more aggressive computational method-backbone grafting of linear motifs-that transplants the backbone and side chains of linear functional motifs onto scaffold proteins. To test this method, we first used side-chain grafting to design new 2F5 epitope scaffolds with improved biophysical characteristics. We then independently transplanted the 2F5 epitope onto three of the same parent scaffolds using the newly developed backbone grafting procedure. Crystal structures of side-chain and backbone grafting designs showed close agreement with both the computational models and the desired epitope structure. In two cases, backbone grafting scaffolds bound antibody 2F5 with 30- and 9-fold higher affinity than corresponding side-chain grafting designs. These results demonstrate that flexible backbone methods for epitope grafting can significantly improve binding affinities over those achieved by fixed backbone methods alone. Backbone grafting of linear motifs is a general method to transplant functional motifs when backbone remodeling of the target scaffold is necessary.


Asunto(s)
Secuencias de Aminoácidos/inmunología , Afinidad de Anticuerpos/inmunología , Epítopos/química , Epítopos/inmunología , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Antígenos Heterófilos/química , Antígenos Heterófilos/inmunología , Sitios de Unión de Anticuerpos , Anticuerpos ampliamente neutralizantes , Simulación por Computador , Cristalografía por Rayos X/métodos , Anticuerpos Anti-VIH , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia
10.
Science ; 334(6054): 373-6, 2011 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-22021856

RESUMEN

The manipulation of protein backbone structure to control interaction and function is a challenge for protein engineering. We integrated computational design with experimental selection for grafting the backbone and side chains of a two-segment HIV gp120 epitope, targeted by the cross-neutralizing antibody b12, onto an unrelated scaffold protein. The final scaffolds bound b12 with high specificity and with affinity similar to that of gp120, and crystallographic analysis of a scaffold bound to b12 revealed high structural mimicry of the gp120-b12 complex structure. The method can be generalized to design other functional proteins through backbone grafting.


Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , Ingeniería de Proteínas , Algoritmos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antígenos CD4/metabolismo , Biología Computacional , Simulación por Computador , Cristalografía por Rayos X , Epítopos/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Resonancia por Plasmón de Superficie
11.
J Neuroimmunol ; 182(1-2): 124-34, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17182110

RESUMEN

Interactions between PD-1 and its two differentially expressed ligands, PD-L1 and PD-L2, attenuate T cell activation and effector function. To determine the role of these molecules in autoimmune disease of the CNS, PD-1-/-, PD-L1-/- and PD-L2-/- mice were generated and immunized to induce experimental autoimmune encephalomyelitis (EAE). PD-1-/- and PD-L1-/- mice developed more severe EAE than wild type and PD-L2-/- mice. Consistent with this, PD-1-/- and PD-L1-/- cells produced elevated levels of the pro-inflammatory cytokines IFN-gamma, TNF, IL-6 and IL-17. These results demonstrate that interactions between PD-1/PD-L1, but not PD-1/PDL-2, are crucial in attenuating T cell responses in EAE.


Asunto(s)
Antígenos de Diferenciación/metabolismo , Antígeno B7-1/metabolismo , Encefalomielitis Autoinmune Experimental/fisiopatología , Glicoproteínas de Membrana/metabolismo , Péptidos/metabolismo , Animales , Antígeno B7-H1 , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Glicoproteínas/inmunología , Humanos , Interferón gamma/biosíntesis , Interleucina-17/biosíntesis , Interleucina-6/biosíntesis , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Activación de Linfocitos , Glicoproteínas de Membrana/deficiencia , Ratones , Ratones Noqueados , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos/inmunología , Péptidos/deficiencia , Proteína 2 Ligando de Muerte Celular Programada 1 , Receptor de Muerte Celular Programada 1 , Índice de Severidad de la Enfermedad , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis
12.
J Exp Med ; 202(6): 841-51, 2005 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-16172261

RESUMEN

Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2alpha (cPLA2alpha), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2alpha-/- mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2alpha+/- mice, whereas the lesions in cPLA2alpha-/- mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2alpha-/- mice compared with cPLA2alpha+/- mice, which indicates that cPLA2alpha plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2alpha-/- mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2alpha also plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2alpha-/- mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2alpha-/- mice susceptible to EAE. Our data indicate that cPLA2alpha plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype.


Asunto(s)
Diferenciación Celular/inmunología , Citosol/enzimología , Encefalomielitis Autoinmune Experimental/enzimología , Encefalomielitis Autoinmune Experimental/genética , Fosfolipasas A/deficiencia , Células TH1/inmunología , Animales , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Fosfolipasas A2 Grupo IV , Inmunidad Innata/genética , Inmunofenotipificación , Interleucina-12/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , Fosfolipasas A/genética , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Médula Espinal/inmunología , Médula Espinal/patología , Células TH1/citología
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