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1.
Immunity ; 55(9): 1710-1724.e8, 2022 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-35944529

RESUMEN

Human metapneumovirus (hMPV) is a leading cause of acute lower respiratory tract infections in high-risk populations, yet there are no vaccines or anti-viral therapies approved for the prevention or treatment of hMPV-associated disease. Here, we used a high-throughput single-cell technology to interrogate memory B cell responses to the hMPV fusion (F) glycoprotein in young adult and elderly donors. Across all donors, the neutralizing antibody response was primarily directed to epitopes expressed on both pre- and post-fusion F conformations. However, we identified rare, highly potent broadly neutralizing antibodies that recognize pre-fusion-specific epitopes and structurally characterized an antibody that targets a site of vulnerability at the pre-fusion F trimer apex. Additionally, monotherapy with neutralizing antibodies targeting three distinct antigenic sites provided robust protection against lower respiratory tract infection in a small animal model. This study provides promising monoclonal antibody candidates for passive immunoprophylaxis and informs the rational design of hMPV vaccine immunogens.


Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Metapneumovirus , Infecciones del Sistema Respiratorio , Anciano , Animales , Epítopos , Glicoproteínas , Humanos , Proteínas Virales de Fusión , Adulto Joven
2.
Int J Mol Sci ; 25(10)2024 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-38791536

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects various mammalian species, with farmed minks experiencing the highest number of outbreaks. In Spain, we analyzed 67 whole genome sequences and eight spike sequences from 18 outbreaks, identifying four distinct lineages: B.1, B.1.177, B.1.1.7, and AY.98.1. The potential risk of transmission to humans raises crucial questions about mutation accumulation and its impact on viral fitness. Sequencing revealed numerous not-lineage-defining mutations, suggesting a cumulative mutation process during the outbreaks. We observed that the outbreaks were predominantly associated with different groups of mutations rather than specific lineages. This clustering pattern by the outbreaks could be attributed to the rapid accumulation of mutations, particularly in the ORF1a polyprotein and in the spike protein. Notably, the mutations G37E in NSP9, a potential host marker, and S486L in NSP13 were detected. Spike protein mutations may enhance SARS-CoV-2 adaptability by influencing trimer stability and binding to mink receptors. These findings provide valuable insights into mink coronavirus genetics, highlighting both host markers and viral transmission dynamics within communities.


Asunto(s)
COVID-19 , Genoma Viral , Visón , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , COVID-19/virología , COVID-19/epidemiología , COVID-19/transmisión , Animales , SARS-CoV-2/genética , SARS-CoV-2/fisiología , España/epidemiología , Visón/virología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Adaptación al Huésped/genética , Humanos , Brotes de Enfermedades , Pandemias , Filogenia , Secuenciación Completa del Genoma
3.
Infection ; 2023 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-37898587

RESUMEN

PURPOSE: We aimed to assess IgG antibodies against the SARS-CoV-2 spike protein (anti-SARS-CoV-2 S IgG) in vaccinated mothers and their infants at delivery and 2-3 months of age. METHODS: We conducted a prospective study on mothers who received at least one dose of the COVID-19 vaccine (Pfizer-BNT162b2, Moderna mRNA-1273, or Oxford-AstraZeneca ChAdOx1-S) during pregnancy and on their infants. The baseline was at the time of delivery (n = 93), and the end of follow-up was 2 to 3 months post-partum (n = 53). Serum anti-SARS-CoV-2 S IgG titers and ACE2 binding inhibition levels were quantified by immunoassays. RESULTS: Mothers and infants had high anti-SARS-CoV-2 S IgG titers against the B.1 lineage at birth. However, while antibody titers were maintained at 2-3 months post-partum in mothers, they decreased significantly in infants (p < 0.001). Positive and significant correlations were found between anti-SARS-CoV-2 S IgG titers and ACE2-binding inhibition levels in mothers and infants at birth and 2-3 months post-partum (r > 0.8, p < 0.001). Anti-S antibodies were also quantified for the Omicron variant at 2-3 months post-partum. The antibody titers against Omicron were significantly lower in mothers and infants than those against B.1 (p < 0.001). Again, a positive correlation was observed for Omicron between IgG titers and ACE2-binding inhibition both in mothers (r = 0.818, p < 0.001) and infants (r = 0.386, p < 0.005). Previous SARS-CoV-2 infection and COVID-19 vaccination near delivery positively impacted anti-SARS-CoV-2 S IgG levels. CONCLUSIONS: COVID-19 mRNA vaccines induce high anti-SARS-CoV-2 S titers in pregnant women, which can inhibit the binding of ACE2 to protein S and are efficiently transferred to the fetus. However, there was a rapid decrease in antibody levels at 2 to 3 months post-partum, particularly in infants.

4.
Birth ; 50(2): 418-427, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-35802776

RESUMEN

INTRODUCTION: Pregnant women are vulnerable to severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. Neutralizing antibodies against the SARS-CoV-2 spike (S) protein protect from severe disease. This study analyzes the antibody titers to SARS-CoV-2 S protein in pregnant women and their newborns at delivery, and six months later. METHODS: We conducted a prospective study on pregnant women with confirmed SARS-CoV-2 infection and newborns. Antibody (IgG, IgM, and IgA) titers were determined using immunoassays in serum and milk samples. An angiotensin-converting enzyme 2 (ACE2) receptor-binding inhibition assay to the S protein was performed on the same serum and milk samples. RESULTS: At birth, antibodies to SARS-CoV-2 spike protein were detected in 81.9% of mothers' sera, 78.9% of cord blood samples, and 63.2% of milk samples. Symptomatic women had higher antibody titers (IgG, IgM, and IgA) than the asymptomatic ones (P < 0.05). At six months postpartum, IgG levels decreased drastically in children's serum (P < 0.001) but remained high in mothers' serum. Antibody titers correlated positively with its capacity to inhibit the ACE2-spike protein interaction at baseline in maternal sera (R2  = 0.203; P < 0.001), cord sera (R2  = 0.378; P < 0.001), and milk (R2  = 0.564; P < 0.001), and at six months in maternal sera (R2  = 0.600; P < 0.001). CONCLUSIONS: High antibody levels against SARS-CoV-2 spike protein were found in most pregnant women. Due to the efficient transfer of IgG to cord blood and high IgA titers in breast milk, neonates may be passively immunized to SARS-CoV-2 infection. Our findings could guide newborn management and maternal vaccination policies.


Asunto(s)
COVID-19 , Complicaciones Infecciosas del Embarazo , Recién Nacido , Embarazo , Femenino , Niño , Humanos , Madres , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2 , Estudios Prospectivos , SARS-CoV-2 , Inmunoglobulina A , Inmunoglobulina G , Inmunoglobulina M
5.
Euro Surveill ; 28(49)2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-38062945

RESUMEN

To advance our understanding of respiratory syncytial virus (RSV) impact through genomic surveillance, we describe two PCR-based sequencing systems, (i) RSVAB-WGS for generic whole-genome sequencing and (ii) RSVAB-GF, which targets major viral antigens, G and F, and is used as a complement for challenging cases with low viral load. These methods monitor RSV genetic diversity to inform molecular epidemiology, vaccine effectiveness and treatment strategies, contributing also to the standardisation of surveillance in a new era of vaccines.


Asunto(s)
Infecciones por Virus Sincitial Respiratorio , Vacunas contra Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Humanos , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Infecciones por Virus Sincitial Respiratorio/epidemiología , Proteínas Virales de Fusión/genética , Vacunas contra Virus Sincitial Respiratorio/genética , Virus Sincitial Respiratorio Humano/genética , Genómica , Secuenciación Completa del Genoma , Anticuerpos Antivirales
6.
J Intern Med ; 291(2): 232-240, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34611927

RESUMEN

BACKGROUND: Anti-SARS-CoV-2 S antibodies prevent viral replication. Critically ill COVID-19 patients show viral material in plasma, associated with a dysregulated host response. If these antibodies influence survival and viral dissemination in ICU-COVID patients is unknown. PATIENTS/METHODS: We studied the impact of anti-SARS-CoV-2 S antibodies levels on survival, viral RNA-load in plasma, and N-antigenaemia in 92 COVID-19 patients over ICU admission. RESULTS: Frequency of N-antigenaemia was >2.5-fold higher in absence of antibodies. Antibodies correlated inversely with viral RNA-load in plasma, representing a protective factor against mortality (adjusted HR [CI 95%], p): (S IgM [AUC ≥ 60]: 0.44 [0.22; 0.88], 0.020); (S IgG [AUC ≥ 237]: 0.31 [0.16; 0.61], <0.001). Viral RNA-load in plasma and N-antigenaemia predicted increased mortality: (N1-viral load [≥2.156 copies/ml]: 2.25 [1.16; 4.36], 0.016); (N-antigenaemia: 2.45 [1.27; 4.69], 0.007). CONCLUSIONS: Low anti-SARS-CoV-2 S antibody levels predict mortality in critical COVID-19. Our findings support that these antibodies contribute to prevent systemic dissemination of SARS-CoV-2.


Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/sangre , COVID-19 , COVID-19/inmunología , COVID-19/mortalidad , Enfermedad Crítica , Humanos , ARN Viral/sangre , SARS-CoV-2
7.
Int J Mol Sci ; 23(6)2022 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-35328398

RESUMEN

The B and T lymphocytes of the adaptive immune system are important for the control of most viral infections, including COVID-19. Identification of epitopes recognized by these cells is fundamental for understanding how the immune system detects and removes pathogens, and for antiviral vaccine design. Intriguingly, several cross-reactive T lymphocyte epitopes from SARS-CoV-2 with other betacoronaviruses responsible for the common cold have been identified. In addition, antibodies that cross-recognize the spike protein, but not the nucleoprotein (N protein), from different betacoronavirus have also been reported. Using a consensus of eight bioinformatic methods for predicting B-cell epitopes and the collection of experimentally detected epitopes for SARS-CoV and SARS-CoV-2, we identified four surface-exposed, conserved, and hypothetical antigenic regions that are exclusive of the N protein. These regions were analyzed using ELISA assays with two cohorts: SARS-CoV-2 infected patients and pre-COVID-19 samples. Here we describe four epitopes from SARS-CoV-2 N protein that are recognized by the humoral response from multiple individuals infected with COVID-19, and are conserved in other human coronaviruses. Three of these linear surface-exposed sequences and their peptide homologs in SARS-CoV-2 and HCoV-OC43 were also recognized by antibodies from pre-COVID-19 serum samples, indicating cross-reactivity of antibodies against coronavirus N proteins. Different conserved human coronaviruses (HCoVs) cross-reactive B epitopes against SARS-CoV-2 N protein are detected in a significant fraction of individuals not exposed to this pandemic virus. These results have potential clinical implications.


Asunto(s)
Proteínas de la Nucleocápside de Coronavirus/inmunología , Coronavirus Humano OC43/inmunología , Reacciones Cruzadas/inmunología , Mapeo Epitopo/métodos , Epítopos de Linfocito B/inmunología , SARS-CoV-2/inmunología , Adulto , Secuencia de Aminoácidos , COVID-19/inmunología , COVID-19/virología , Estudios de Cohortes , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/genética , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/fisiología , Reacciones Cruzadas/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos de Linfocito B/metabolismo , Células HEK293 , Personal de Salud/estadística & datos numéricos , Humanos , Dominios Proteicos , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Homología de Secuencia de Aminoácido , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología
8.
Angew Chem Int Ed Engl ; 61(28): e202203662, 2022 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-35507573

RESUMEN

The development of versatile and sensitive biotools to quantify specific SARS-CoV-2 immunoglobulins in SARS-CoV-2 infected and non-infected individuals, built on the surface of magnetic microbeads functionalized with nucleocapsid (N) and in-house expressed recombinant spike (S) proteins is reported. Amperometric interrogation of captured N- and S-specific circulating total or individual immunoglobulin (Ig) isotypes (IgG, IgM, and IgA), subsequently labelled with HRP-conjugated secondary antibodies, was performed at disposable single or multiplexed (8×) screen-printed electrodes using the HQ/HRP/H2 O2 system. The obtained results using N and in-house expressed S ectodomains of five SARS-CoV-2 variants of concern (including the latest Delta and Omicron) allow identification of vulnerable populations from those with natural or acquired immunity, monitoring of infection, evaluation of vaccine efficiency, and even identification of the variant responsible for the infection.


Asunto(s)
Técnicas Biosensibles , COVID-19 , Anticuerpos Antivirales , COVID-19/diagnóstico , Prueba de COVID-19 , Humanos , Inmunidad , Inmunoglobulina G , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus
9.
J Biol Chem ; 294(49): 18600-18612, 2019 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-31640987

RESUMEN

During the late phase of the HIV-1 replication cycle, the viral Gag polyproteins are targeted to the plasma membrane for assembly. The Gag-membrane interaction is mediated by binding of Gag's N-terminal myristoylated matrix (MA) domain to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The viral envelope (Env) glycoprotein is then recruited to the assembly sites and incorporated into budding particles. Evidence suggests that Env incorporation is mediated by interactions between Gag's MA domain and the cytoplasmic tail of the gp41 subunit of Env (gp41CT). MA trimerization appears to be an obligatory step for this interaction. Insufficient production of a recombinant MA trimer and unavailability of a biologically relevant membrane system have been barriers to detailed structural and biophysical characterization of the putative MA-gp41CT-membrane interactions. Here, we engineered a stable recombinant HIV-1 MA trimer construct by fusing a foldon domain (FD) of phage T4 fibritin to the MA C terminus. Results from NMR experiments confirmed that the FD attachment does not adversely alter the MA structure. Employing hydrogen-deuterium exchange MS, we identified an MA-MA interface in the MA trimer that is implicated in Gag assembly and Env incorporation. Utilizing lipid nanodiscs as a membrane mimetic, we show that the MA trimer binds to membranes 30-fold tighter than does the MA monomer and that incorporation of PI(4,5)P2 and phosphatidylserine enhances the binding of MA to nanodiscs. These findings advance our understanding of a fundamental mechanism in HIV-1 assembly and provide a template for investigating the interaction of MA with gp41CT.


Asunto(s)
VIH-1/metabolismo , Ensamble de Virus/fisiología , Calorimetría , Membrana Celular/metabolismo , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/metabolismo , Espectroscopía de Resonancia Magnética , Fosfatidilserinas/metabolismo , Unión Proteica
10.
Anal Bioanal Chem ; 411(26): 6801-6811, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31422432

RESUMEN

Mycotoxins can be found as natural contaminants in many foods and feeds, and owing to their toxic effects, it is essential to detect them before they enter the food chain. An interesting approach for the analysis of mycotoxins by competitive immunoassays is the use of epitope-mimicking peptides, or mimotopes, which can replace the toxin conjugates traditionally used in such assays. Mimotopes can be selected from phage-displayed peptide libraries even without any prior knowledge of the antibody-antigen interaction, and after identifying the target specific clones, individual clones can be efficiently amplified in bacteria and used directly in the immunoassay. Following such approach, we have previously selected and identified a dodecapeptide which functions as a mimotope for the mycotoxin fumonisin B1. In this work, we present the development and comparison of various immunoassays based on this mimotope, named A2, which has been used in the phage-displayed format in which it was selected, but also as a fluorescent recombinant fusion protein or as a synthetic peptide. The highest sensitivity was obtained with a magnetic bead-based assay using the synthetic peptide and enzymatic detection which provided a detection limit of 0.029 ng mL-1. Analysis of the binding kinetics by surface plasmon resonance (SPR) further reinforced the suitability of the synthetic peptide for the competitive immunoassays, as this mimotope showed a slightly lower affinity for the target antibody in comparison with the recombinant fusion protein. Graphical abstract.


Asunto(s)
Fumonisinas/análisis , Inmunoensayo/métodos , Micotoxinas/análisis , Biblioteca de Péptidos , Péptidos/química , Técnicas de Visualización de Superficie Celular/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Límite de Detección , Proteínas Luminiscentes/química , Proteínas Recombinantes de Fusión/química , Resonancia por Plasmón de Superficie/métodos
11.
PLoS Pathog ; 12(9): e1005859, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27611367

RESUMEN

Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.


Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Metapneumovirus/inmunología , Proteínas Virales de Fusión/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/química , Antígenos Virales/genética , Reacciones Cruzadas , Cristalografía por Rayos X , Femenino , Ingeniería Genética , Humanos , Metapneumovirus/genética , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Conformación Molecular , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/inmunología , Alineación de Secuencia , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética
12.
J Virol ; 90(11): 5485-5498, 2016 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-27009962

RESUMEN

UNLABELLED: Human respiratory syncytial virus (hRSV) vaccine development has received new impetus from structure-based studies of its main protective antigen, the fusion (F) glycoprotein. Three soluble forms of F have been described: monomeric, trimeric prefusion, and trimeric postfusion. Most human neutralizing antibodies recognize epitopes found exclusively in prefusion F. Although prefusion F induces higher levels of neutralizing antibodies than does postfusion F, postfusion F can also induce protection against virus challenge in animals. However, the immunogenicity and protective efficacy of the three forms of F have not hitherto been directly compared. Hence, BALB/c mice were immunized with a single dose of the three proteins adjuvanted with CpG and challenged 4 weeks later with virus. Serum antibodies, lung virus titers, weight loss, and pulmonary pathology were evaluated after challenge. Whereas small amounts of postfusion F were sufficient to protect mice, larger amounts of monomeric and prefusion F proteins were required for protection. However, postfusion and monomeric F proteins were associated with more pathology after challenge than was prefusion F. Antibodies induced by all doses of prefusion F, in contrast to other F protein forms, reacted predominantly with the prefusion F conformation. At high doses, prefusion F also induced the highest titers of neutralizing antibodies, and all mice were protected, yet at low doses of the immunogen, these antibodies neutralized virus poorly, and mice were not protected. These findings should be considered when developing new hRSV vaccine candidates. IMPORTANCE: Protection against hRSV infection is afforded mainly by neutralizing antibodies, which recognize mostly epitopes found exclusively in the viral fusion (F) glycoprotein trimer, folded in its prefusion conformation, i.e., before activation for membrane fusion. Although prefusion F is able to induce high levels of neutralizing antibodies, highly stable postfusion F (found after membrane fusion) is also able to induce neutralizing antibodies and protect against infection. In addition, a monomeric form of hRSV F that shares epitopes with prefusion F was recently reported. Since each of the indicated forms of hRSV F may have advantages and disadvantages for the development of safe and efficacious subunit vaccines, a direct comparison of the immunogenic properties and protective efficacies of the different forms of hRSV F was made in a mouse model. The results obtained show important differences between the noted immunogens that should be borne in mind when considering the development of hRSV vaccines.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Virus Sincitiales Respiratorios/química , Virus Sincitiales Respiratorios/inmunología , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/inmunología , Adyuvantes Inmunológicos , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Relación Dosis-Respuesta Inmunológica , Epítopos/inmunología , Femenino , Humanos , Inmunización , Inmunogenicidad Vacunal , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Oligodesoxirribonucleótidos/inmunología , Conformación Proteica , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/aislamiento & purificación , Proteínas Virales de Fusión/administración & dosificación
13.
PLoS Pathog ; 11(7): e1005035, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26161532

RESUMEN

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.


Asunto(s)
Anticuerpos Neutralizantes/ultraestructura , Anticuerpos Antivirales/ultraestructura , Epítopos de Linfocito B/ultraestructura , Virus Sincitiales Respiratorios/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Línea Celular , Cromatografía en Gel , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Citometría de Flujo , Glicoproteínas/química , Glicoproteínas/inmunología , Glicoproteínas/ultraestructura , Humanos , Estructura Cuaternaria de Proteína , Resonancia por Plasmón de Superficie
14.
Antimicrob Agents Chemother ; 60(11): 6498-6509, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27550346

RESUMEN

ALX-0171 is a trivalent Nanobody derived from monovalent Nb017 that binds to antigenic site II of the human respiratory syncytial virus (hRSV) fusion (F) glycoprotein. ALX-0171 is about 6,000 to 10,000 times more potent than Nb017 in neutralization tests with strains of hRSV antigenic groups A and B. To explore the effect of this enhanced neutralization on escape mutant selection, viruses resistant to either ALX-0171 or Nb017 were isolated after serial passage of the hRSV Long strain in the presence of suboptimal concentrations of the respective Nanobodies. Resistant viruses emerged notably faster with Nb017 than with ALX-0171 and in both cases contained amino acid changes in antigenic site II of hRSV F. Detailed binding and neutralization analyses of these escape mutants as well as previously described mutants resistant to certain monoclonal antibodies (MAbs) offered a comprehensive description of site II mutations which are relevant for neutralization by MAbs and Nanobodies. Notably, ALX-0171 showed a sizeable neutralization potency with most escape mutants, even with some of those selected with the Nanobody, and these findings make ALX-0171 an attractive antiviral for treatment of hRSV infections.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/farmacología , Antígenos Virales/inmunología , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Proteínas Virales de Fusión/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/química , Anticuerpos Antivirales/aislamiento & purificación , Antígenos Virales/química , Antígenos Virales/genética , Camélidos del Nuevo Mundo , Línea Celular Tumoral , Células Epiteliales/virología , Epítopos/química , Epítopos/inmunología , Humanos , Evasión Inmune/genética , Sueros Inmunes/química , Modelos Moleculares , Mutación , Pruebas de Neutralización , Unión Proteica , Estructura Secundaria de Proteína , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/aislamiento & purificación , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunología
15.
Antimicrob Agents Chemother ; 60(1): 6-13, 2016 01.
Artículo en Inglés | MEDLINE | ID: mdl-26438495

RESUMEN

Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated in vitro neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitiales Respiratorios/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Proteínas Virales de Fusión/antagonistas & inhibidores , Administración por Inhalación , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Antivirales/inmunología , Antivirales/metabolismo , Antivirales/farmacología , Femenino , Expresión Génica , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/virología , Masculino , Modelos Moleculares , Cavidad Nasal/efectos de los fármacos , Cavidad Nasal/inmunología , Cavidad Nasal/virología , Pruebas de Neutralización , Palivizumab/biosíntesis , Palivizumab/inmunología , Palivizumab/farmacología , Pichia/genética , Pichia/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/patología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/inmunología , Virus Sincitiales Respiratorios/patogenicidad , Sigmodontinae , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/inmunología , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunología , Carga Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos
16.
J Virol ; 88(20): 11802-10, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25078705

RESUMEN

Respiratory syncytial virus (RSV) is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. There remains an unmet vaccine need despite decades of research. Insufficient potency, homogeneity, and stability of previous RSV fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation, producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from prefusion trimers to postfusion trimers. Using RSV F constructs with mutated furin cleavage sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires specific cleavage between F1 and F2 for self-association and rearrangement into stable postfusion trimers. The uncleaved RSV F monomer is folded and homogenous and displays at least two key RSV-neutralizing epitopes shared between the prefusion and postfusion conformations. Unlike the cleaved trimer, the uncleaved monomer binds the prefusion-specific monoclonal antibody D25 and human neutralizing immunoglobulins that do not bind to postfusion F. These observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and is a potential prefusion subunit vaccine candidate. Importance: RSV is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical trial using a formalin-inactivated RSV virus made disease, following RSV infection, more severe. Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before and after virus entry (prefusion and postfusion conformations, respectively). Key neutralization epitopes of prefusion and postfusion RSV F have been identified, and a number of current vaccine development efforts are focused on generating easily produced subunit antigens that retain these epitopes. Here we show that a simple modification in the F ectodomain results in a homogeneous protein that retains critical prefusion neutralizing epitopes. These results improve our understanding of RSV F protein folding and structure and can guide further vaccine design efforts.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Epítopos/inmunología , Virus Sincitiales Respiratorios/inmunología , Humanos , Proteolisis
17.
Proc Natl Acad Sci U S A ; 109(8): 3089-94, 2012 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-22323598

RESUMEN

Human respiratory syncytial virus (hRSV) is the most important viral agent of pediatric respiratory infections worldwide. The only specific treatment available today is a humanized monoclonal antibody (Palivizumab) directed against the F glycoprotein, administered prophylactically to children at very high risk of severe hRSV infections. Palivizumab, as most anti-F antibodies so far described, recognizes an epitope that is shared by the two conformations in which hRSV_F can fold, the metastable prefusion form and the highly stable postfusion conformation. We now describe a unique class of antibodies specific for the prefusion form of this protein that account for most of the neutralizing activity of either a rabbit serum raised against a vaccinia virus recombinant expressing hRSV_F or a human Ig preparation (Respigam), which was used for prophylaxis before Palivizumab. These antibodies therefore offer unique possibilities for immune intervention against hRSV, and their production should be assessed in trials of hRSV vaccines.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Infecciones por Virus Sincitial Respiratorio/terapia , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales de Fusión/inmunología , Secuencia de Aminoácidos , Animales , Humanos , Inmunización , Datos de Secuencia Molecular , Estabilidad Proteica , Conejos , Proteínas Recombinantes/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Vaccinia/inmunología , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/ultraestructura
18.
J Gen Virol ; 95(Pt 5): 1033-1042, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24531414

RESUMEN

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283-6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.


Asunto(s)
Antígenos Virales/genética , Antígenos Virales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Epítopos/genética , Epítopos/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Mutación Missense , ARN Viral/genética , Análisis de Secuencia de ADN , España
19.
Subcell Biochem ; 68: 467-87, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23737062

RESUMEN

Viruses are intracellular parasites that hijack the cellular machinery for their own replication. Therefore, an obligatory step in the virus life cycle is the delivery of the viral genome inside the cell. Enveloped viruses (i.e., viruses with a lipid envelope) use a two-step procedure to release their genetic material into the cell: (i) they first bind to specific surface receptors of the target cell membrane and then, (ii) they fuse the viral and cell membranes. This last step may occur at the cell surface or after internalization of the virus particle by endocytosis or by some other route (e.g., macropinocytosis). Remarkably, the virus-cell membrane fusion process goes essentially along the same intermediate steps as other membrane fusions that occur for instance in vesicular fusion at the nerve synapsis or cell-cell fusion in yeast mating. Specialized viral proteins, fusogens, promote virus-cell membrane fusion. The viral fusogens experience drastic structural rearrangements during fusion, liberating the energy required to overcome the repulsive forces that prevent spontaneous fusion of the two membranes. This chapter describes the different types of viral fusogens and their mode of action, as are currently known.


Asunto(s)
Membrana Celular/metabolismo , Fusión de Membrana/fisiología , Internalización del Virus , Virus/metabolismo , Animales , Humanos , Replicación Viral
20.
Viruses ; 16(6)2024 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-38932235

RESUMEN

Following an interseasonal rise in mainly pediatric respiratory syncytial virus (RSV) cases in Germany in 2021, an exceptionally high number of adult cases was observed in the subsequent respiratory season of 2022/2023. The aim of this study was to compare the clinical presentation of RSV infections in the pre- and post-SARS-CoV-2 pandemic periods. Additionally, the local epidemiology of the RSV fusion protein was analyzed at a molecular genetic and amino acid level. RSV detections in adults peaked in calendar week 1 of 2023, 8 weeks earlier than the earliest peak observed in the three pre-pandemic seasons. Although the median age of the adult patients was not different (66.5 vs. 65 years), subtle differences between both periods regarding comorbidities and the clinical presentation of RSV cases were noted. High rates of comorbidities prevailed; however, significantly lower numbers of patients with a history of lung transplantation (p = 0.009), chronic kidney disease (p = 0.013), and immunosuppression (p = 0.038) were observed in the 2022/2023 season. In contrast, significantly more lower respiratory tract infections (p < 0.001), in particular in the form of pneumonia (p = 0.015) and exacerbations of obstructive lung diseases (p = 0.008), were detected. An ICU admission was noted for 23.7% of all patients throughout the study period. Sequence analysis of the fusion protein gene revealed a close phylogenetic relatedness, regardless of the season of origin. However, especially for RSV-B, an accumulation of amino acid point substitutions was noted, including in antigenic site Ø. The SARS-CoV-2 pandemic had a tremendous impact on the seasonality of RSV, and the introduction of new vaccination and immunization strategies against RSV warrants further epidemiologic studies of this important pathogen.


Asunto(s)
COVID-19 , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Estaciones del Año , Centros de Atención Terciaria , Proteínas Virales de Fusión , Humanos , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones por Virus Sincitial Respiratorio/virología , Proteínas Virales de Fusión/genética , Virus Sincitial Respiratorio Humano/genética , Alemania/epidemiología , Femenino , Centros de Atención Terciaria/estadística & datos numéricos , Anciano , Masculino , Persona de Mediana Edad , COVID-19/epidemiología , COVID-19/virología , Adulto , SARS-CoV-2/genética , Epidemiología Molecular , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/virología , Anciano de 80 o más Años , Adulto Joven , Filogenia
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