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1.
PLoS Pathog ; 18(7): e1010691, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35862475

RESUMEN

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel ß-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human ß-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human ß-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.


Asunto(s)
COVID-19 , Glicoproteína de la Espiga del Coronavirus , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales , COVID-19/terapia , Vacunas contra la COVID-19 , Humanos , Ratones , SARS-CoV-2 , Pérdida de Peso
2.
J Virol ; 96(8): e0166821, 2022 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-35343783

RESUMEN

Binding to the receptor, CD4, drives the pretriggered, "closed" (state-1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer into more "open" conformations (states 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the state-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize states 2/3. HIV-1 Env metastability has created challenges for defining the state-1 structure and developing immunogens mimicking this labile conformation. The availability of functional state-1 Envs that can be efficiently cross-linked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association, and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4, and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered state-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly cross-linkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state. IMPORTANCE The development of an efficient vaccine is critical for combating HIV-1 infection worldwide. However, the instability of the pretriggered shape (state 1) of the viral envelope glycoprotein (Env) makes it difficult to raise neutralizing antibodies against HIV-1. Here, by introducing multiple changes in Env, we derived two HIV-1 Env variants that are enriched in state 1 and can be efficiently cross-linked to maintain this shape. These Env complexes are more stable in detergent, assisting their purification. Thus, our study provides a path to a better characterization of the native pretriggered Env, which should assist vaccine development.


Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Detergentes , Glicoproteínas/química , Glicoproteínas/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/prevención & control , VIH-1/química , VIH-1/genética , VIH-1/inmunología , Humanos , Lisina , Conformación Proteica , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología
3.
Proc Natl Acad Sci U S A ; 116(26): 13036-13041, 2019 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-31189602

RESUMEN

Human cytomegalovirus (HCMV) causes severe disease in infants and immunocompromised people. There is no approved HCMV vaccine, and vaccine development strategies are complicated by evidence of both persistent infection and reinfection of people with prior immunity. The greatest emphasis has been placed on reducing transmission to seronegative pregnant women to prevent vertical transmission and its potentially severe sequelae. Increasing evidence suggests that the earliest host-HCMV interactions establish conditions for viral persistence, including evasion of host immune responses to the virus. Using a nonhuman primate model of HCMV infection, we show that rhesus macaques immunized against viral interleukin-10 (IL-10) manifest delayed rhesus cytomegalovirus (RhCMV) acquisition and altered immune responses to the infection when it does occur. Among animals with the greatest antiviral IL-10-neutralizing activity, the timing of RhCMV seroconversion was delayed by an average of 12 weeks. After acquisition, such animals displayed an antibody response to the new infection, which peaked as expected after 2 weeks but then declined rapidly. In contrast, surprisingly, vaccination with glycoprotein B (gB) protein had no discernible impact on these outcomes. Our results demonstrate that viral IL-10 is a key regulator of successful host immune responses to RhCMV. Viral IL-10 is, therefore, an important target for vaccine strategies against cytomegalovirus (CMV). Furthermore, given the immunoregulatory function of viral IL-10, targeting this protein may prove synergistic with other vaccine therapies and targets. Our study also provides additional evidence that the earliest host-CMV interactions can have a significant impact on the nature of persistent infection.


Asunto(s)
Antígenos Virales/inmunología , Infecciones por Citomegalovirus/prevención & control , Vacunas contra Citomegalovirus/farmacología , Citomegalovirus/inmunología , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Interleucina-10/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/administración & dosificación , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/transmisión , Infecciones por Citomegalovirus/virología , Vacunas contra Citomegalovirus/inmunología , Vacunas contra Citomegalovirus/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Mucosa , Inmunogenicidad Vacunal , Interleucina-10/administración & dosificación , Macaca mulatta , Embarazo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Proteínas del Envoltorio Viral/administración & dosificación , Esparcimiento de Virus/inmunología
4.
Lupus ; 29(9): 1095-1105, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32611267

RESUMEN

BACKGROUND/OBJECTIVE: Type-I interferons contribute to pathogenesis in systemic lupus erythematosus, including nephritis. Interferons consist of a family of 16 proteins yet are often characterized in patients without knowledge of the specific interferon subtypes involved. Different interferons may function in the kidneys, and other organs, relative to what is often measured in patient blood. Moreover, antibodies to interferons may potentially modulate systemic or organ-specific interferon activity. The aim of this study was to characterize global interferon activity levels and identify autoantibodies to the 12 interferon α subtypes in patient serum and urine. METHODS: Interferon activity levels in serum and urine were measured using an interferon bioassay. Anti-interferon and anti-cytokine autoantibodies were measured by ELISA. Serum and urine samples were also characterized for their ability to neutralize the biological activity of exogenously added interferons. RESULTS: Serum interferon activity was increased in 62% of systemic lupus erythematosus patient samples, relative to healthy donor controls, whereas binding interferon α autoantibodies to at least one interferon α subtype were found in 68% of the samples evaluated. High Systemic Lupus Erythematosus Disease Activity Index scores were significantly (p = 0.001) associated with patient samples containing interferon α autoantibodies to three or more interferon α subtypes in their serum. Interferon α autoantibodies that potently block interferon activity were rare (∼5% of samples), but collectively bound to all 12 interferon α subtypes. Urine interferon activity and interferon α autoantibody profiles did not correlate with their serum counterparts, suggesting immune responses in systemic lupus erythematosus kidneys can be distinct from those measured in serum. Analysis of autoantibodies to 15 additional cytokines in serum identified higher frequencies of granulocyte-macrophage colony-stimulating factor and interleukin 17A autoantibodies, suggesting these signaling pathways may potentially contribute, with interferons, to systemic lupus erythematosus pathogenesis. CONCLUSIONS: The measurement of autoantibodies to multiple interferon subtypes in serum and urine may provide an alternative method for following interferon-mediated systemic lupus erythematosus disease activity. The results suggest autoantibodies might be used for patient monitoring and/or identifying additional cytokine signaling pathways that are functioning in different systemic lupus erythematosus patients.


Asunto(s)
Autoanticuerpos/sangre , Autoanticuerpos/orina , Interferón Tipo I/inmunología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/orina , Estudios de Casos y Controles , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Lupus Eritematoso Sistémico/inmunología
5.
J Biol Chem ; 291(1): 447-61, 2016 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-26546677

RESUMEN

Exogenous cytokine therapy can induce systemic toxicity, which might be prevented by activating endogenously produced cytokines in local cell niches. Here we developed antibody-based activators of cytokine signaling (AcCS), which recognize cytokines only when they are bound to their cell surface receptors. AcCS were developed for type I interferons (IFNs), which induce cellular activities by binding to cell surface receptors IFNAR1 and IFNAR2. As a potential alternative to exogenous IFN therapy, AcCS were shown to potentiate the biological activities of natural IFNs by ∼100-fold. Biochemical and structural characterization demonstrates that the AcCS stabilize the IFN-IFNAR2 binary complex by recognizing an IFN-induced conformational change in IFNAR2. Using IFN mutants that disrupt IFNAR1 binding, AcCS were able to enhance IFN antiviral potency without activating antiproliferative responses. This suggests AcCS can be used to manipulate cytokine signaling for basic science and possibly for therapeutic applications.


Asunto(s)
Citocinas/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Receptores de Citocinas/inmunología , Transducción de Señal , Antivirales/química , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Fragmentos de Inmunoglobulinas/farmacología , Interferón-alfa/farmacología , Cinética , Mutación/genética , Fosforilación , Conformación Proteica , Receptor de Interferón alfa y beta/química , Receptor de Interferón alfa y beta/metabolismo , Reproducibilidad de los Resultados , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos
6.
J Virol ; 90(21): 9920-9930, 2016 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-27558431

RESUMEN

There is accumulating evidence that the viral interleukin-10 (vIL-10) ortholog of both human and rhesus cytomegalovirus (HCMV and RhCMV, respectively) suppresses the functionality of cell types that are critical to contain virus dissemination and help shape long-term immunity during the earliest virus-host interactions. In particular, exposure of macrophages, peripheral blood mononuclear cells, monocyte-derived dendritic cells, and plasmacytoid dendritic cells to vIL-10 suppresses multiple effector functions including, notably, those that link innate and adaptive immune responses. Further, vaccination of RhCMV-uninfected rhesus macaques with nonfunctional forms of RhCMV vIL-10 greatly restricted parameters of RhCMV infection following RhCMV challenge of the vaccinees. Vaccinees exhibited significantly reduced shedding of RhCMV in saliva and urine following RhCMV challenge compared to shedding in unvaccinated controls. Based on the evidence that vIL-10 is critical during acute infection, the role of vIL-10 during persistent infection was analyzed in rhesus macaques infected long term with RhCMV to determine whether postinfection vaccination against vIL-10 could change the virus-host balance. RhCMV-seropositive macaques, which shed RhCMV in saliva, were vaccinated with nonfunctional RhCMV vIL-10, and shedding levels of RhCMV in saliva were evaluated. Following robust increases in vIL-10-binding and vIL-10-neutralizing antibodies, shedding levels of RhCMV modestly declined, consistent with the interpretation that vIL-10 may play a functional role during persistent infection. However, a more significant association was observed between the levels of cellular IL-10 secreted in peripheral blood mononuclear cells exposed to RhCMV antigens and shedding of RhCMV in saliva. This result implies that RhCMV persistence is associated with the induction of cellular IL-10 receptor-mediated signaling pathways. IMPORTANCE: Human health is adversely impacted by viruses that establish lifelong infections that are often accompanied with increased morbidity and mortality (e.g., infections with HIV, hepatitis C virus, or human cytomegalovirus). A longstanding but unfulfilled goal has been to develop postinfection vaccine strategies that could "reboot" the immune system of an infected individual in ways that would enable the infected host to develop immune responses that clear reservoirs of persistent virus infection, effectively curing the host of infection. This concept was evaluated in rhesus macaques infected long term with rhesus cytomegalovirus by repeatedly immunizing infected animals with nonfunctional versions of the rhesus cytomegalovirus-encoded viral interleukin-10 immune-modulating protein. Following vaccine-mediated boosting of antibody titers to viral interleukin-10, there was modest evidence for increased immunological control of the virus following vaccination. More significantly, data were also obtained that indicated that rhesus cytomegalovirus is able to persist due to upregulation of the cellular interleukin-10 signaling pathway.


Asunto(s)
Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/virología , Citomegalovirus/metabolismo , Interleucina-10/metabolismo , Macaca mulatta/metabolismo , Macaca mulatta/virología , Transducción de Señal/fisiología , Animales , Interacciones Huésped-Patógeno/fisiología , Inmunización Secundaria/métodos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Vacunación/métodos , Proteínas Virales/metabolismo , Esparcimiento de Virus/fisiología
7.
Proc Natl Acad Sci U S A ; 109(31): 12704-9, 2012 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-22802649

RESUMEN

Interleukin 20 (IL-20) is a pleotropic IL-10 family cytokine that protects epithelial surfaces from pathogens. However, dysregulated IL-20 signaling is implicated in several human pathologies including psoriasis, rheumatoid arthritis, atherosclerosis, and osteoporosis. IL-20, and related cytokines IL-19 and IL-24, designated IL-20 subfamily cytokines (IL-20SFCs), induce cellular responses through an IL-20R1/IL-20R2 (type I) receptor heterodimer, whereas IL-20 and IL-24 also signal through the IL-22R1/IL-20R2 (type II) receptor complex. The crystal structure of the IL-20/IL-20R1/IL-20R2 complex reveals how type I and II complexes discriminate cognate from noncognate ligands. The structure also defines how the receptor-cytokine interfaces are affinity tuned to allow distinct signaling through a receptor complex shared by three different ligands. Our results provide unique insights into the complexity of IL-20SFC signaling that may be critical in the design of mechanistic-based inhibitors of IL-20SFC-mediated inflammatory disease.


Asunto(s)
Interleucinas/química , Receptores de Interleucina/química , Artritis Reumatoide/metabolismo , Aterosclerosis/metabolismo , Cristalografía por Rayos X , Humanos , Interleucinas/metabolismo , Osteoporosis/metabolismo , Unión Proteica , Multimerización de Proteína/fisiología , Estructura Cuaternaria de Proteína , Psoriasis/metabolismo , Receptores de Interleucina/metabolismo , Relación Estructura-Actividad
8.
J Virol ; 87(21): 11323-31, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23946461

RESUMEN

Identification of immune correlates of protection for viral vaccines is complicated by multiple factors, but there is general consensus on the importance of antibodies that neutralize viral attachment to susceptible cells. Development of new viral vaccines has mostly followed this neutralizing antibody paradigm, but as a recent clinical trial of human cytomegalovirus (HCMV) vaccination demonstrated, this singular approach can yield limited protective efficacy. Since HCMV devotes >50% of its coding capacity to proteins that modulate host immunity, it is hypothesized that expansion of vaccine targets to include this part of the viral proteome will disrupt viral natural history. HCMV and rhesus cytomegalovirus (RhCMV) each encode an ortholog to the cellular interleukin-10 (cIL-10) cytokine: cmvIL-10 and rhcmvIL10, respectively. Despite extensive sequence divergence from their host's cIL-10, each viral IL-10 retains nearly identical functionality to cIL-10. Uninfected rhesus macaques were immunized with engineered, nonfunctional rhcmvIL-10 variants, which were constructed by site-directed mutagenesis to abolish binding to the cIL-10 receptor. Vaccinees developed antibodies that neutralized rhcmvIL-10 function with no cross-neutralization of cIL-10. Following subcutaneous RhCMV challenge, the vaccinees exhibited both reduced RhCMV replication locally at the inoculation site and systemically and significantly reduced RhCMV shedding in bodily fluids compared to controls. Attenuation of RhCMV infection by rhcmvIL-10 vaccination argues that neutralization of viral immunomodulation may be a new vaccine paradigm for HCMV by expanding potential vaccine targets.


Asunto(s)
Vacunas contra Citomegalovirus/inmunología , Interleucina-10/inmunología , Vacunación/métodos , Proteínas Virales/inmunología , Factores de Virulencia/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/prevención & control , Vacunas contra Citomegalovirus/administración & dosificación , Vacunas contra Citomegalovirus/genética , Modelos Animales de Enfermedad , Interleucina-10/genética , Macaca mulatta , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Proteínas Virales/genética , Factores de Virulencia/genética , Replicación Viral
9.
FEBS J ; 290(13): 3422-3435, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37014961

RESUMEN

Monoclonal antibodies that retain neutralizing activity against multiple coronavirus (CoV) lineages and variants of concern (VoC) must be developed to protect against future pandemics. These broadly neutralizing MAbs (BNMAbs) may be used as therapeutics and/or to assist in the rational design of vaccines that induce BNMAbs. 1249A8 is a BNMAb that targets the stem helix (SH) region of CoV spike (S) protein and neutralizes Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) original strain, delta, and omicron VoC, Severe Acute Respiratory Syndrome CoV (SARS-CoV), and Middle East Respiratory Syndrome CoV (MERS-CoV). To understand its mechanism of action, the crystal structure of 1249A8 bound to a MERS-CoV SH peptide was determined at 2.1 Å resolution. BNMAb 1249A8 mimics the SARS-CoV-2 S loop residues 743-749, which interacts with the N-terminal end of the SH helix in the S post-fusion conformation. The conformation of 1249A8-bound SH is distinct from the SH conformation observed in the post-fusion SARS-CoV-2 S structure, suggesting 1249A8 disrupts the secondary structure and refolding events required for CoV post-fusion S to initiate membrane fusion and ultimately infection. This study provides novel insights into the neutralization mechanisms of SH-targeting CoV BNMAbs that may inform vaccine development and the design of optimal BNMAb therapeutics.


Asunto(s)
COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , Anticuerpos Neutralizantes , Epítopos , Anticuerpos Antivirales , Anticuerpos Monoclonales , SARS-CoV-2
10.
Front Immunol ; 12: 691715, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34149735

RESUMEN

Severe acute respiratory syndrome coronavirus-2 (SAR-CoV-2) causes coronavirus disease 2019 (COVID19) that is responsible for short and long-term disease, as well as death, in susceptible hosts. The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) protein binds to cell surface angiotensin converting enzyme type-II (ACE2) to initiate viral attachment and ultimately viral pathogenesis. The SARS-CoV-2 S RBD is a major target of neutralizing antibodies (NAbs) that block RBD - ACE2 interactions. In this report, NAb-RBD binding epitopes in the protein databank were classified as C1, C1D, C2, C3, or C4, using a RBD binding profile (BP), based on NAb-specific RBD buried surface area and used to predict the binding epitopes of a series of uncharacterized NAbs. Naturally occurring SARS-CoV-2 RBD sequence variation was also quantified to predict NAb binding sensitivities to the RBD-variants. NAb and ACE2 binding studies confirmed the NAb classifications and determined whether the RBD variants enhanced ACE2 binding to promote viral infectivity, and/or disrupted NAb binding to evade the host immune response. Of 9 single RBD mutants evaluated, K417T, E484K, and N501Y disrupted binding of 65% of the NAbs evaluated, consistent with the assignment of the SARS-CoV-2 P.1 Japan/Brazil strain as a variant of concern (VoC). RBD variants E484K and N501Y exhibited ACE2 binding equivalent to a Wuhan-1 reference SARS-CoV-2 RBD. While slightly less disruptive to NAb binding, L452R enhanced ACE2 binding affinity. Thus, the L452R mutant, associated with the SARS-CoV-2 California VoC (B.1.427/B.1.429-California), has evolved to enhance ACE2 binding, while simultaneously disrupting C1 and C2 NAb classes. The analysis also identified a non-overlapping antibody pair (1213H7 and 1215D1) that bound to all SARS-CoV-2 RBD variants evaluated, representing an excellent therapeutic option for treatment of SARS-CoV-2 WT and VoC strains.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Epítopos de Linfocito B/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Sitios de Unión de Anticuerpos , Epítopos de Linfocito B/química , Humanos , Mutación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/inmunología , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología
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