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1.
Cell ; 176(6): 1432-1446.e11, 2019 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-30827685

RESUMEN

The presence of DNA in the cytosol of mammalian cells is an unusual event that is often associated with genotoxic stress or viral infection. The enzyme cGAS is a sensor of cytosolic DNA that induces interferon and inflammatory responses that can be protective or pathologic, depending on the context. Along with other cytosolic innate immune receptors, cGAS is thought to diffuse throughout the cytosol in search of its DNA ligand. Herein, we report that cGAS is not a cytosolic protein but rather localizes to the plasma membrane via the actions of an N-terminal phosphoinositide-binding domain. This domain interacts selectively with PI(4,5)P2, and cGAS mutants defective for lipid binding are mislocalized to the cytosolic and nuclear compartments. Mislocalized cGAS induces potent interferon responses to genotoxic stress, but weaker responses to viral infection. These data establish the subcellular positioning of a cytosolic innate immune receptor as a mechanism that governs self-nonself discrimination.


Asunto(s)
Membrana Celular/fisiología , Nucleotidiltransferasas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Citosol/fisiología , ADN Viral/genética , Femenino , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata/fisiología , Interferones/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Nucleotidiltransferasas/fisiología , Fosfatidilinositol 4,5-Difosfato/fisiología , Fosfatidilinositoles , Unión Proteica , Transducción de Señal/inmunología
2.
Cell ; 161(6): 1280-92, 2015 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-26004070

RESUMEN

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.


Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Linfocitos B/inmunología , Antígenos CD4/metabolismo , Regiones Determinantes de Complementariedad , Epítopos de Linfocito B , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia
3.
Nature ; 509(7498): 55-62, 2014 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-24590074

RESUMEN

Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/química , Proteínas gp160 de Envoltorio del VIH/inmunología , Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/aislamiento & purificación , Afinidad de Anticuerpos/genética , Afinidad de Anticuerpos/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Sitios de Unión/inmunología , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Linaje de la Célula , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Mapeo Epitopo , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Evolución Molecular , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/aislamiento & purificación , Infecciones por VIH/inmunología , VIH-1/química , VIH-1/inmunología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Neutralización , Estructura Terciaria de Proteína , Hipermutación Somática de Inmunoglobulina/genética
4.
J Virol ; 90(1): 76-91, 2016 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-26468542

RESUMEN

UNLABELLED: The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of 12 V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C-infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al., Nature 509:55-62, 2014, http://dx.doi.org/10.1038/nature13036). Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight were isolated using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed a 10-fold greater neutralization potency than previously published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among the viruses neutralized, the median 50% inhibitory concentration was 0.001 µg/ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, the CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent. IMPORTANCE: Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C-infected donor. Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed a 10-fold greater neutralization potency than previously described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: the median 50% inhibitory concentration was 0.001 µg/ml. Our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Reacciones Cruzadas , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/aislamiento & purificación , Mapeo Epitopo , Femenino , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/aislamiento & purificación , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Concentración 50 Inhibidora , Datos de Secuencia Molecular , Análisis de Secuencia de ADN
5.
mBio ; 12(2)2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33849978

RESUMEN

Mammalian cells detect microbial molecules known as pathogen-associated molecular patterns (PAMPs) as indicators of potential infection. Upon PAMP detection, diverse defensive responses are induced by the host, including those that promote inflammation and cell-intrinsic antimicrobial activities. Host-encoded molecules released from dying or damaged cells, known as damage-associated molecular patterns (DAMPs), also induce defensive responses. Both DAMPs and PAMPs are recognized for their inflammatory potential, but only the latter are well established to stimulate cell-intrinsic host defense. Here, we report a class of DAMPs that engender an antiviral state in human epithelial cells. These DAMPs include oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine), PGPC (1-palmitoyl-2-glutaryl phosphatidylcholine), and POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine], oxidized lipids that are naturally released from dead or dying cells. Exposing cells to these DAMPs prior to vesicular stomatitis virus (VSV) infection limits viral replication. Mechanistically, these DAMPs prevent viral entry, thereby limiting the percentage of cells that are productively infected and consequently restricting viral load. We found that the antiviral actions of oxidized lipids are distinct from those mediated by the PAMP Poly I:C, in that the former induces a more rapid antiviral response without the induction of the interferon response. These data support a model whereby interferon-independent defensive activities can be induced by DAMPs, which may limit viral replication before PAMP-mediated interferon responses are induced. This antiviral activity may impact viruses that disrupt interferon responses in the oxygenated environment of the lung, such as influenza virus and SARS-CoV-2.IMPORTANCE In this work, we explored how a class of oxidized lipids, spontaneously created during tissue damage and unprogrammed cell lysis, block the earliest events in RNA virus infection in the human epithelium. This gives us novel insight into the ways that we view infection models, unveiling a built-in mechanism to slow viral growth that neither engages the interferon response nor is subject to known viral antagonism. These oxidized phospholipids act prior to infection, allowing time for other, better-known innate immune mechanisms to take effect. This discovery broadens our understanding of host defenses, introducing a soluble factor that alters the cellular environment to protect from RNA virus infection.


Asunto(s)
Alarminas/farmacología , Antivirales/farmacología , Virus ARN/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Células A549 , Muerte Celular/efectos de los fármacos , Humanos , Inmunidad Innata , Interferones/genética , Interferones/metabolismo , Cinética , Moléculas de Patrón Molecular Asociado a Patógenos/farmacología , Fosfatidilcolinas/farmacología , Virus ARN/fisiología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Vesiculovirus/efectos de los fármacos , Vesiculovirus/fisiología , Carga Viral
6.
Nat Med ; 22(12): 1465-1469, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27820604

RESUMEN

For broad protection against infection by viruses such as influenza or HIV, vaccines should elicit antibodies that bind conserved viral epitopes, such as the receptor-binding site (RBS). RBS-directed antibodies have been described for both HIV and influenza virus, and the design of immunogens to elicit them is a goal of vaccine research in both fields. Residues in the RBS of influenza virus hemagglutinin (HA) determine a preference for the avian or human receptor, α-2,3-linked sialic acid and α-2,6-linked sialic acid, respectively. Transmission of an avian-origin virus between humans generally requires one or more mutations in the sequences encoding the influenza virus RBS to change the preferred receptor from avian to human, but passage of a human-derived vaccine candidate in chicken eggs can select for reversion to avian receptor preference. For example, the X-181 strain of the 2009 new pandemic H1N1 influenza virus, derived from the A/California/07/2009 isolate and used in essentially all vaccines since 2009, has arginine at position 226, a residue known to confer preference for an α-2,3 linkage in H1 subtype viruses; the wild-type A/California/07/2009 isolate, like most circulating human H1N1 viruses, has glutamine at position 226. We describe, from three different individuals, RBS-directed antibodies that recognize the avian-adapted H1 strain in current influenza vaccines but not the circulating new pandemic 2009 virus; Arg226 in the vaccine-strain RBS accounts for the restriction. The polyclonal sera of the three donors also reflect this preference. Therefore, when vaccines produced from strains that are never passaged in avian cells become widely available, they may prove more capable of eliciting RBS-directed, broadly neutralizing antibodies than those produced from egg-adapted viruses, extending the established benefits of current seasonal influenza immunizations.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Ácidos Siálicos/inmunología , Animales , Pollos , Cristalización , Perros , Huevos , Humanos , Inmunogenicidad Vacunal/inmunología , Gripe Aviar/virología , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Células Plasmáticas/inmunología , Estructura Terciaria de Proteína
7.
Nat Struct Mol Biol ; 23(1): 81-90, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26689967

RESUMEN

Broadly neutralizing antibodies (bNAbs) against HIV-1 Env V1V2 arise in multiple donors. However, atomic-level interactions had previously been determined only with antibodies from a single donor, thus making commonalities in recognition uncertain. Here we report the cocrystal structure of V1V2 with antibody CH03 from a second donor and model Env interactions of antibody CAP256-VRC26 from a third donor. These V1V2-directed bNAbs used strand-strand interactions between a protruding antibody loop and a V1V2 strand but differed in their N-glycan recognition. Ontogeny analysis indicated that protruding loops develop early, and glycan interactions mature over time. Altogether, the multidonor information suggested that V1V2-directed bNAbs form an 'extended class', for which we engineered ontogeny-specific antigens: Env trimers with chimeric V1V2s that interacted with inferred ancestor and intermediate antibodies. The ontogeny-based design of vaccine antigens described here may provide a general means for eliciting antibodies of a desired class.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Línea Celular , Cristalografía por Rayos X , VIH-1/química , VIH-1/inmunología , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica
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