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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.
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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 SecuenciaRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1007431.].
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[This corrects the article DOI: 10.1371/journal.ppat.1008026.].
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The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.
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Vacunas contra el SIDA/inmunología , Anticuerpos ampliamente neutralizantes/biosíntesis , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/biosíntesis , Anticuerpos Anti-VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/química , Vacunas contra el SIDA/genética , Sustitución de Aminoácidos , Afinidad de Anticuerpos , Sitios de Unión , Antígenos CD4/metabolismo , Diseño de Fármacos , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Células HEK293 , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/patogenicidad , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/inmunología , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Inducing broad spectrum neutralizing antibodies against challenging pathogens such as HIV-1 is a major vaccine design goal, but may be hindered by conformational instability within viral envelope glycoproteins (Env). Chemical cross-linking is widely used for vaccine antigen stabilization, but how this process affects structure, antigenicity and immunogenicity is poorly understood and its use remains entirely empirical. We have solved the first cryo-EM structure of a cross-linked vaccine antigen. The 4.2 Å structure of HIV-1 BG505 SOSIP soluble recombinant Env in complex with a CD4 binding site-specific broadly neutralizing antibody (bNAb) Fab fragment reveals how cross-linking affects key properties of the trimer. We observed density corresponding to highly specific glutaraldehyde (GLA) cross-links between gp120 monomers at the trimer apex and between gp120 and gp41 at the trimer interface that had strikingly little impact on overall trimer conformation, but critically enhanced trimer stability and improved Env antigenicity. Cross-links were also observed within gp120 at sites associated with the N241/N289 glycan hole that locally modified trimer antigenicity. In immunogenicity studies, the neutralizing antibody response to cross-linked trimers showed modest but significantly greater breadth against a global panel of difficult-to-neutralize Tier-2 heterologous viruses. Moreover, the specificity of autologous Tier-2 neutralization was modified away from the N241/N289 glycan hole, implying a novel specificity. Finally, we have investigated for the first time T helper cell responses to next-generation soluble trimers, and report on vaccine-relevant immunodominant responses to epitopes within BG505 that are modified by cross-linking. Elucidation of the structural correlates of a cross-linked viral glycoprotein will allow more rational use of this methodology for vaccine design, and reveals a strategy with promise for eliciting neutralizing antibodies needed for an effective HIV-1 vaccine.
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VIH-1/química , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo/inmunología , Reactivos de Enlaces Cruzados , Microscopía por Crioelectrón , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/química , Antígenos VIH/inmunología , Antígenos VIH/ultraestructura , Interacciones Huésped-Patógeno/inmunología , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Conformación Proteica , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Conejos , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/ultraestructuraRESUMEN
Broadly neutralizing antibody (bnAb) induction is a high priority for effective HIV-1 vaccination. VRC01-class bnAbs that target the CD4 binding site (CD4bs) of trimeric HIV-1 envelope (Env) glycoprotein spikes are particularly attractive to elicit because of their extraordinary breadth and potency of neutralization in vitro and their ability to protect against infection in animal models. Glycans bordering the CD4bs impede the binding of germline-reverted forms of VRC01-class bnAbs and therefore constitute a barrier to early events in initiating the correct antibody lineages. Deleting a subset of these glycans permits Env antigen binding but not virus neutralization, suggesting that additional barriers impede germline-reverted VRC01-class antibody binding to functional Env trimers. We investigated the requirements for functional Env trimer engagement of VRC01-class naïve B cell receptors by using virus neutralization and germline-reverted antibodies as surrogates for the interaction. Targeted deletion of a subset of N-glycans bordering the CD4bs, combined with Man5 enrichment of remaining N-linked glycans that are otherwise processed into larger complex-type glycans, rendered HIV-1 426c Env-pseudotyped virus (subtype C, transmitted/founder) highly susceptible to neutralization by near germline forms of VRC01-class bnAbs. Neither glycan modification alone rendered the virus susceptible to neutralization. The potency of neutralization in some cases rivaled the potency of mature VRC01 against wildtype viruses. Neutralization by the germline-reverted antibodies was abrogated by the known VRC01 resistance mutation, D279K. These findings improve our understanding of the restrictions imposed by glycans in eliciting VRC01-class bnAbs and enable a neutralization-based strategy to monitor vaccine-elicited early precursors of this class of bnAbs.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Linfocitos B/inmunología , Sitios de Unión , Anticuerpos ampliamente neutralizantes , Antígenos CD4/inmunología , Epítopos/inmunología , Glicosilación , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Seropositividad para VIH , VIH-1/inmunología , Humanos , Polisacáridos/inmunología , Polisacáridos/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
UNLABELLED: Major neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability. Stabilized soluble trimers such as BG505 SOSIP.664 mimic the structure of virion-associated Env but nevertheless sample different conformational states. Here we demonstrate that treating BG505 SOSIP.664 trimers with glutaraldehyde or a heterobifunctional cross-linker introduces additional stability with relatively modest effects on antigenicity. Thus, most broadly neutralizing antibody (bNAb) epitopes were preserved after cross-linking, whereas the binding of most weakly or nonneutralizing antibodies (non-NAb) was reduced. Cross-linking stabilized all Env conformers present within a mixed population, and individual conformers could be isolated by bNAb affinity chromatography. Both positive selection of cross-linked conformers using the quaternary epitope-specific bNAbs PGT145, PGT151, and 3BC315 and negative selection with non-NAbs against the V3 region enriched for trimer populations with improved antigenicity for bNAbs. Similar results were obtained using the clade B B41 SOSIP.664 trimer. The cross-linking method may, therefore, be useful for countering the natural conformational heterogeneity of some HIV-1 Env proteins and, by extrapolation, also vaccine immunogens from other pathogens. IMPORTANCE: The development of a vaccine to induce protective antibodies against HIV-1 is of primary public health importance. Recent advances in immunogen design have provided soluble recombinant envelope glycoprotein trimers with near-native morphology and antigenicity. However, these trimers are conformationally flexible, potentially reducing B-cell recognition of neutralizing antibody epitopes. Here we show that chemical cross-linking increases trimer stability, reducing binding of nonneutralizing antibodies while largely maintaining neutralizing antibody binding. Cross-linking followed by positive or negative antibody affinity selection of individual stable conformational variants further improved the antigenic and morphological characteristics of the trimers. This approach may be generally applicable to HIV-1 Env and also to other conformationally flexible pathogen antigens.
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Antígenos VIH/inmunología , Antígenos VIH/metabolismo , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Anticuerpos Neutralizantes/inmunología , Reactivos de Enlaces Cruzados/metabolismo , Anticuerpos Anti-VIH/inmunología , HumanosRESUMEN
Macrophage infection is considered to play an important role in HIV-1 pathogenesis and persistence. Using a primary cell-based coculture model, we show that monocyte-derived macrophages (MDM) efficiently transmit a high-multiplicity HIV-1 infection to autologous CD4(+) T cells through a viral envelope glycoprotein (Env) receptor- and actin-dependent virological synapse (VS), facilitated by interactions between ICAM-1 and LFA-1. Virological synapse (VS)-mediated transmission by MDM results in high levels of T cell HIV-1 integration and is 1 to 2 orders of magnitude more efficient than cell-free infection. This mode of cell-to-cell transmission is broadly susceptible to the activity of CD4 binding site (CD4bs) and glycan or glycopeptide epitope-specific broadly neutralizing monoclonal antibodies (bNMAbs) but shows resistance to bNMAbs targeting the Env gp41 subunit membrane-proximal external region (MPER). These data define for the first time the structure and function of the macrophage-to-T cell VS and have important implications for bNMAb activity in HIV-1 prophylaxis and therapy. IMPORTANCE The ability of HIV-1 to move directly between contacting immune cells allows efficient viral dissemination with the potential to evade antibody attack. Here, we show that HIV-1 spreads from infected macrophages to T cells via a structure called a virological synapse that maintains extended contact between the two cell types, allowing transfer of multiple infectious events to the T cell. This process allows the virus to avoid neutralization by a class of antibody targeting the gp41 subunit of the envelope glycoproteins. These results have implications for viral spread in vivo and the specificities of neutralizing antibody elicited by antibody-based vaccines.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/transmisión , Evasión Inmune/inmunología , Sinapsis Inmunológicas/virología , Macrófagos/inmunología , Análisis de Varianza , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/virología , Cartilla de ADN/genética , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Luciferasas , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Macrófagos/virología , Microscopía Confocal , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Imagen de Lapso de TiempoRESUMEN
Polyethyleneimine (PEI) is an organic polycation used extensively as a gene and DNA vaccine delivery reagent. Although the DNA targeting activity of PEI is well documented, its immune activating activity is not. We recently reported that PEI has robust mucosal adjuvanticity when administered intranasally with glycoprotein antigens. Here, we show that PEI has strong immune activating activity after systemic delivery. PEI administered subcutaneously with viral glycoprotein (HIV-1 gp140) enhanced antigen-specific serum IgG production in the context of mixed Th1/Th2-type immunity. PEI elicited higher titers of both antigen binding and neutralizing antibodies than alum in mice and rabbits and induced an increased proportion of antibodies reactive with native antigen. In an intraperitoneal model, PEI recruited neutrophils followed by monocytes to the site of administration and enhanced antigen uptake by antigen-presenting cells. The Th bias was modulated by PEI activation of the Nlrp3 inflammasome; however its global adjuvanticity was unchanged in Nlrp3-deficient mice. When coformulated with CpG oligodeoxynucleotides, PEI adjuvant potency was synergistically increased and biased toward a Th1-type immune profile. Taken together, these data support the use of PEI as a versatile systemic adjuvant platform with particular utility for induction of secondary structure-reactive antibodies against glycoprotein antigens.
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Adyuvantes Inmunológicos , Antígenos/inmunología , Glicoproteínas/inmunología , Polietileneimina , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos/inmunología , Células Presentadoras de Antígenos/inmunología , Quimiotaxis de Leucocito , Citocinas/biosíntesis , Inmunización , Ratones , Ratones Noqueados , Oligodesoxirribonucleótidos/inmunología , Polietileneimina/administración & dosificación , Conejos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Efferocytic clearance of apoptotic cells in general, and T cells in particular, is required for tissue and immune homeostasis. Transmembrane mucins are extended glycoproteins highly expressed in the cell glycocalyx that function as a barrier to phagocytosis. Whether and how mucins may be regulated during cell death to facilitate efferocytic corpse clearance is not well understood. Here we show that normal and transformed human T cells express a subset of mucins which are rapidly and selectively removed from the cell surface during apoptosis. This process is mediated by the ADAM10 sheddase, the activity of which is associated with XKR8-catalyzed flipping of phosphatidylserine to the outer leaflet of the plasma membrane. Mucin clearance enhances uptake of apoptotic T cells by macrophages, confirming mucins as an enzymatically-modulatable barrier to efferocytosis. Together these findings demonstrate a glycocalyx regulatory pathway with implications for therapeutic intervention in the clearance of normal and transformed apoptotic T cells.
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Eferocitosis , Mucinas , Humanos , Linfocitos T/metabolismo , Apoptosis , Fagocitosis , Proteína ADAM10/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del AmiloideRESUMEN
Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although ß-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical ß-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)-regardless of CDRH3 length-limits cross-reactivity against other ß-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the 'germline-guided reverse engineering' of such drugs away from unwanted immune responses.
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Antibacterianos , Animales , Ratones , Antibacterianos/farmacología , Antibacterianos/inmunología , Inmunoglobulina G/inmunología , Penicilina G/inmunología , Penicilina G/química , Linfocitos B/inmunología , Penicilinas/inmunología , Penicilinas/química , Femenino , Reacciones Cruzadas/inmunología , Cristalografía por Rayos XRESUMEN
The focus of most current HIV-1 vaccine development is on antibody-based approaches. This is because certain antibody responses correlated with protection from HIV-1 acquisition in the RV144 phase III trial, and because a series of potent and broad spectrum neutralizing antibodies have been isolated from infected individuals. Taken together, these two findings suggest ways forward to develop a neutralizing antibody-based vaccine. However, understanding of the correlates of protection from disease in HIV-1 and other infections strongly suggests that we should not ignore CTL-based research. Here we review recent progress in the field and highlight the challenges implicit in HIV-1 vaccine design and some potential solutions.
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Vacunas contra el SIDA/inmunología , Síndrome de Inmunodeficiencia Adquirida/prevención & control , VIH-1/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/aislamiento & purificación , Síndrome de Inmunodeficiencia Adquirida/inmunología , Anticuerpos Neutralizantes/sangre , Ensayos Clínicos como Asunto , Descubrimiento de Drogas/tendencias , Anticuerpos Anti-VIH/sangre , Humanos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Direct cell-cell spread of Human Immunodeficiency Virus type-1 (HIV-1) at the virological synapse (VS) is an efficient mode of dissemination between CD4(+) T cells but the mechanisms by which HIV-1 proteins are directed towards intercellular contacts is unclear. We have used confocal microscopy and electron tomography coupled with functional virology and cell biology of primary CD4(+) T cells from normal individuals and patients with Chediak-Higashi Syndrome and report that the HIV-1 VS displays a regulated secretion phenotype that shares features with polarized secretion at the T cell immunological synapse (IS). Cell-cell contact at the VS re-orientates the microtubule organizing center (MTOC) and organelles within the HIV-1-infected T cell towards the engaged target T cell, concomitant with polarization of viral proteins. Directed secretion of proteins at the T cell IS requires specialized organelles termed secretory lysosomes (SL) and we show that the HIV-1 envelope glycoprotein (Env) localizes with CTLA-4 and FasL in SL-related compartments and at the VS. Finally, CD4(+) T cells that are disabled for regulated secretion are less able to support productive cell-to-cell HIV-1 spread. We propose that HIV-1 hijacks the regulated secretory pathway of CD4(+) T cells to enhance its dissemination.
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Linfocitos T CD4-Positivos/metabolismo , VIH-1/fisiología , Sinapsis/fisiología , Proteínas Virales/metabolismo , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Síndrome de Chediak-Higashi/fisiopatología , Síndrome de Chediak-Higashi/virología , Humanos , Lisosomas/fisiología , Microscopía Confocal , Centro Organizador de los Microtúbulos/fisiología , Proteínas Qa-SNARE/fisiología , Vías Secretoras , Internalización del VirusRESUMEN
Oxidative stress is widespread and entwined with pathological processes, yet its linkage to adaptive immunity remains elusive. Reactive carbonyl (RC) adduction, a common feature of oxidative stress, has been shown to target proteins to the adaptive immune system. Because aldehydes are important mediators of carbonylation, we explored the immunomodulatory properties of model Ags modified by common bioactive aldehyde by-products of oxidative stress: 4-hydroxy-2-nonenal, malondialdehyde, and glycolaldehyde. Ag modification with all three aldehydes resulted in Ag-specific IgG1-dominated responses in adjuvant-free murine immunizations in an RC-dependent manner. The central role of RCs was confirmed, as their reduction into nonreactive groups abrogated all adaptive responses, despite the presence of other well-known aldehyde-driven adducts such as N(ε)-carboxymethyllysine and glycolaldehyde-pyridine. Moreover, Ag-specific Ab responses robustly correlated with the extent of RC adduction, regardless of the means of their generation. T cell responses mirrored the Th2-biased Ab isotypes by Ag-specific splenocyte production of IL-4, IL-5, and IL-13, but not IFN-γ. The RC-induced Th2 response was in sharp contrast to that induced by Th1/Th2 balanced or Th1-biasing adjuvants and was maintained in a range of mouse strains. In vitro studies revealed that RC adduction enhanced Ag presentation with Th2 polarization in the absence of conventional dendritic cell activation. Taken together, these data implicate commonly occurring RC as an important oxidation-derived Th2 immunomodulatory damage-associated molecular pattern with potentially important roles in health and disease.
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Aldehídos/inmunología , Antígenos/inmunología , Estrés Oxidativo/inmunología , Células Th2/inmunología , Aldehídos/metabolismo , Animales , Antígenos/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones SCID , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/metabolismoRESUMEN
Heat, oxidation and exposure to aldehydes create reactive carbonyl groups on proteins, targeting antigens to scavenger receptors. Formaldehyde is widely used in making vaccines, but has been associated with atypical enhanced disease during subsequent infection with paramyxoviruses. We show that carbonyl groups on formaldehyde-treated vaccine antigens boost T helper type 2 (T(H)2) responses and enhance respiratory syncytial virus (RSV) disease in mice, an effect partially reversible by chemical reduction of carbonyl groups.
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Formaldehído , Inmunización/efectos adversos , Hipersensibilidad Respiratoria/etiología , Vacunas de Productos Inactivados/efectos adversos , Animales , Antígenos Virales/inmunología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Eosinofilia/etiología , Eosinofilia/patología , Interferón gamma/inmunología , Interleucina-5/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Ovalbúmina/inmunología , Hipersensibilidad Respiratoria/patología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/efectos adversos , Vacunas contra Virus Sincitial Respiratorio/inmunología , Vacunas contra Virus Sincitial Respiratorio/farmacología , Virus Sincitiales Respiratorios/inmunología , Células Th2/inmunología , Factores de Tiempo , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/farmacologíaRESUMEN
As obligate intracellular parasites, viruses must bind to, and enter, permissive host cells in order to gain access to the cellular machinery that is required for their replication. The very large number of mammalian viruses identified to date is reflected in the fact that almost every human and animal cell type is a target for infection by one, or commonly more than one, species of virus. As viruses have adapted to target certain cell types for their propagation, there is exquisite specificity in cellular tropism. This specificity is frequently, but not always, mediated by the first step in the viral replication cycle: attachment of viral surface proteins to receptors expressed on susceptible cells. Viral receptors may be protein, carbohydrate, and/or lipid. Many viruses can use more than one attachment receptor, and indeed may sequentially engage multiple receptors to infect a cell. Thus, it is useful to differentiate between attachment receptors, that simply allow viruses a foothold at the limiting membrane of a cell, and entry receptors that mediate delivery the viral genome into the cytoplasm. For some viruses the attachment factors that promote binding to permissive cells are very well defined, but the sequence of events that triggers viral entry is only now beginning to be understood. For other viruses, despite many efforts, the receptors remain elusive. In this chapter we will confine our review to viruses that infect mammals, with particular focus on human pathogens. We do not intend that this will be an exhaustive overview of viral attachment receptors; instead we will take a number of examples of well-characterized virus-receptor interactions, discuss supporting evidence, and highlight any controversies and uncertainties in the field. We will then conclude with a reflection on general principles of viral attachment, consider some exceptions to these principles, and make some suggestion for future research.
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Receptores Virales/fisiología , Acoplamiento Viral , Animales , Antígenos CD4 , Moléculas de Adhesión Celular/fisiología , Humanos , Integrinas/fisiología , Lectinas Tipo C/fisiología , Ácido N-Acetilneuramínico/fisiología , Receptores de Superficie Celular/fisiología , Internalización del VirusRESUMEN
Chemical cross-linking is used to stabilize protein structures with additional benefits of pathogen and toxin inactivation for vaccine use, but its use has been restricted by the potential for local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for inducing a biological outcome such as the elicitation of antibodies to conformationally sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) to cross-link two soluble Env trimers, selected well-folded trimer species using antibody affinity, and transferred this process to good manufacturing practice (GMP) for experimental medicine use. Cross-linking enhanced trimer stability to biophysical and enzyme attack. Cryo-EM analysis revealed that cross-linking retained the overall structure with root-mean-square deviations (RMSDs) between unmodified and cross-linked Env trimers of 0.4-0.5 Å. Despite this negligible distortion of global trimer structure, we identified individual inter-subunit, intra-subunit, and intra-protomer cross-links. Antigenicity and immunogenicity of the trimers were selectively modified by cross-linking, with cross-linked ConS retaining bnAb binding more consistently than ConM. Thus, the EDC cross-linking process improves trimer stability whilst maintaining protein folding, and is readily transferred to GMP, consistent with the more general use of this approach in protein-based vaccine design.
RESUMEN
Productive infection of macrophages is central to HIV-1 pathogenesis. Newly formed virions bud into a tubular membranous compartment that is contiguous with the plasma membrane. However, little is known about the structure of this compartment and its potential regulation by infection. Here we characterized this compartment in macrophages using electron tomography and electron microscopy with stereology. We found an intricate, interconnected membrane network that constitutes a preexisting physiologic structure in macrophages but which expands in size upon HIV-1 infection. Membranes required for this expansion were apparently derived from preexisting pools of plasma membrane. Physical connections between this compartment and the extracellular milieu were frequently made by tube-like structures of insufficient diameter for virion passage. We conclude that HIV-1 induces the expansion of a complex membranous labyrinth in macrophages in which the virus buds and can be retained, with potential consequences for transmission and immune evasion.
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Compartimento Celular , VIH-1/fisiología , Macrófagos/virología , Ensamble de Virus , Humanos , Microscopía ElectrónicaRESUMEN
Immune tolerance to self-glycans is a host mechanism to avoid autoimmunity, which is exploited by HIV-1 coating its envelope glycoproteins with glycans to evade neutralising antibodies (nAbs). Huettner et al. describe cross-reactivity between Schistosoma mansoni glycans and HIV-1 envelope glycoprotein glycans, suggesting a strategy for induction of HIV-1 nAbs by vaccination.