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1.
Nat Immunol ; 25(6): 1083-1096, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38816616

RESUMEN

Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.


Asunto(s)
Afinidad de Anticuerpos , Linfocitos B , Centro Germinal , Anticuerpos Anti-VIH , VIH-1 , Centro Germinal/inmunología , Animales , Ratones , Humanos , Linfocitos B/inmunología , VIH-1/inmunología , Anticuerpos Anti-VIH/inmunología , Afinidad de Anticuerpos/inmunología , Anticuerpos Neutralizantes/inmunología , Infecciones por VIH/inmunología , Vacunas contra el SIDA/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Técnicas de Sustitución del Gen , Ratones Transgénicos , Anticuerpos ampliamente neutralizantes/inmunología , Ratones Endogámicos C57BL
2.
EMBO J ; 40(2): e105926, 2021 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-33258500

RESUMEN

B-cell receptor (BCR) knock-in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one-step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD-GT8 60mer, a germline-targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class-switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases.


Asunto(s)
Linfocitos B/metabolismo , Sistemas CRISPR-Cas/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Sistemas CRISPR-Cas/inmunología , Línea Celular , Técnicas de Sustitución del Gen/métodos , Centro Germinal/inmunología , Centro Germinal/metabolismo , Células HEK293 , VIH-1/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Receptores de Antígenos de Linfocitos B/inmunología
3.
Proc Natl Acad Sci U S A ; 117(37): 22920-22931, 2020 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-32873644

RESUMEN

Animal models of human antigen-specific B cell receptors (BCRs) generally depend on "inferred germline" sequences, and thus their relationship to authentic naive human B cell BCR sequences and affinities is unclear. Here, BCR sequences from authentic naive human VRC01-class B cells from healthy human donors were selected for the generation of three BCR knockin mice. The BCRs span the physiological range of affinities found in humans, and use three different light chains (VK3-20, VK1-5, and VK1-33) found among subclasses of naive human VRC01-class B cells and HIV broadly neutralizing antibodies (bnAbs). The germline-targeting HIV immunogen eOD-GT8 60mer is currently in clinical trial as a candidate bnAb vaccine priming immunogen. To attempt to model human immune responses to the eOD-GT8 60mer, we tested each authentic naive human VRC01-class BCR mouse model under rare human physiological B cell precursor frequency conditions. B cells with high (HuGL18HL) or medium (HuGL17HL) affinity BCRs were primed, recruited to germinal centers, and they affinity matured, and formed memory B cells. Precursor frequency and affinity interdependently influenced responses. Taken together, these experiments utilizing authentic naive human VRC01-class BCRs validate a central tenet of germline-targeting vaccine design and extend the overall concept of the reverse vaccinology approach to vaccine development.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos/genética , Animales , Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/farmacología , Antígenos CD4/inmunología , Técnicas de Sustitución del Gen/métodos , Centro Germinal/inmunología , Antígenos VIH , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Ratones , Ratones Endogámicos , Ratones Transgénicos , Células Precursoras de Linfocitos B/inmunología , Vacunación/métodos
4.
PLoS Pathog ; 13(2): e1006212, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-28225819

RESUMEN

Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design.


Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Anticuerpos Bloqueadores/química , Anticuerpos Bloqueadores/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Conformación Proteica , Resonancia por Plasmón de Superficie , Difracción de Rayos X
5.
J Am Chem Soc ; 136(13): 5047-56, 2014 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-24611715

RESUMEN

Despite recent advances in genome engineering made possible by the emergence of site-specific endonucleases, there remains a need for tools capable of specifically delivering genetic payloads into the human genome. Hybrid recombinases based on activated catalytic domains derived from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are a class of reagents capable of achieving this. The utility of these enzymes, however, has been constrained by their low overall targeting specificity, largely due to the formation of side-product homodimers capable of inducing off-target modifications. Here, we combine rational design and directed evolution to re-engineer the serine recombinase dimerization interface and generate a recombinase architecture that reduces formation of these undesirable homodimers by >500-fold. We show that these enhanced recombinases demonstrate substantially improved targeting specificity in mammalian cells and achieve rates of site-specific integration similar to those previously reported for site-specific nucleases. Additionally, we show that enhanced recombinases exhibit low toxicity and promote the delivery of the human coagulation factor IX and α-galactosidase genes into endogenous genomic loci with high specificity. These results provide a general means for improving hybrid recombinase specificity by protein engineering and illustrate the potential of these enzymes for basic research and therapeutic applications.


Asunto(s)
Ingeniería de Proteínas/métodos , Recombinasas/química , Recombinasas/genética , Recombinación Genética , Dedos de Zinc , Secuencia de Aminoácidos , Dominio Catalítico , ADN/genética , Evolución Molecular Dirigida/métodos , Factor IX/genética , Genoma Humano , Células HEK293 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinasas/metabolismo , alfa-Galactosidasa/genética
6.
Sci Immunol ; 9(95): eadn0622, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38753808

RESUMEN

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.


Asunto(s)
Anticuerpos ampliamente neutralizantes , Nanopartículas , ARN Mensajero , Animales , Ratones , Humanos , ARN Mensajero/inmunología , ARN Mensajero/genética , Nanopartículas/química , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Lípidos/inmunología , Infecciones por VIH/inmunología , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , VIH-1/inmunología , Femenino , Anticuerpos Monoclonales , Liposomas
7.
Sci Transl Med ; 16(748): eadn0223, 2024 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-38753806

RESUMEN

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.


Asunto(s)
Vacunas contra el SIDA , Anticuerpos Neutralizantes , Anticuerpos Anti-VIH , Animales , Humanos , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Ratones , Vacunación , Inmunización Secundaria , VIH-1/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Anticuerpos ampliamente neutralizantes/inmunología
8.
Science ; 378(6623): eadd6502, 2022 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-36454825

RESUMEN

Broadly neutralizing antibodies (bnAbs) can protect against HIV infection but have not been induced by human vaccination. A key barrier to bnAb induction is vaccine priming of rare bnAb-precursor B cells. In a randomized, double-blind, placebo-controlled phase 1 clinical trial, the HIV vaccine-priming candidate eOD-GT8 60mer adjuvanted with AS01B had a favorable safety profile and induced VRC01-class bnAb precursors in 97% of vaccine recipients with median frequencies reaching 0.1% among immunoglobulin G B cells in blood. bnAb precursors shared properties with bnAbs and gained somatic hypermutation and affinity with the boost. The results establish clinical proof of concept for germline-targeting vaccine priming, support development of boosting regimens to induce bnAbs, and encourage application of the germline-targeting strategy to other targets in HIV and other pathogens.


Asunto(s)
Vacunas contra el SIDA , Anticuerpos ampliamente neutralizantes , Células Germinativas , Anticuerpos Anti-VIH , Infecciones por VIH , Cadenas Pesadas de Inmunoglobulina , Cadenas Ligeras de Inmunoglobulina , Humanos , Adyuvantes Inmunológicos , Vacunas contra el SIDA/inmunología , Anticuerpos ampliamente neutralizantes/genética , Anticuerpos ampliamente neutralizantes/inmunología , Infecciones por VIH/prevención & control , Vacunación , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/inmunología , Células Germinativas/inmunología , Linfocitos B/inmunología , Mutación , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Masculino , Femenino , Adulto
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