RESUMEN
Epitope-targeted HIV vaccine design seeks to focus antibody responses to broadly neutralizing antibody (bnAb) sites by sequential immunization. A chimpanzee simian immunodeficiency virus (SIV) envelope (Env) shares a single bnAb site, the variable loop 2 (V2)-apex, with HIV, suggesting its possible utility in an HIV immunization strategy. Here, we generate a chimpanzee SIV Env trimer, MT145K, which displays selective binding to HIV V2-apex bnAbs and precursor versions, but no binding to other HIV specificities. We determine the structure of the MT145K trimer by cryo-EM and show that its architecture is remarkably similar to HIV Env. Immunization of an HIV V2-apex bnAb precursor Ab-expressing knockin mouse with the chimpanzee MT145K trimer induces HIV V2-specific neutralizing responses. Subsequent boosting with an HIV trimer cocktail induces responses that exhibit some virus cross-neutralization. Overall, the chimpanzee MT145K trimer behaves as expected from design both in vitro and in vivo and is an attractive potential component of a sequential immunization regimen to induce V2-apex bnAbs.
Asunto(s)
Vacunas contra el SIDA/química , Productos del Gen env/química , Virus de la Inmunodeficiencia de los Simios/metabolismo , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Reacciones Antígeno-Anticuerpo , Microscopía por Crioelectrón , Productos del Gen env/genética , Productos del Gen env/inmunología , Productos del Gen env/metabolismo , Glicosilación , Anticuerpos Anti-VIH/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Mutagénesis Sitio-Dirigida , Pan troglodytes/virología , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Ingeniería de Proteínas , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunologíaRESUMEN
Recent efforts toward HIV vaccine development include the design of immunogens that can engage B cell receptors with the potential to affinity mature into broadly neutralizing antibodies (bnAbs). V2-apex bnAbs, which bind a protein-glycan region on HIV envelope glycoprotein (Env) trimer, are among the most broad and potent described. We show here that a rare "glycan hole" at the V2 apex is enriched in HIV isolates neutralized by inferred precursors of prototype V2-apex bnAbs. To investigate whether this feature could focus neutralizing responses onto the apex bnAb region, we immunized wild-type rabbits with soluble trimers adapted from these Envs. Potent autologous tier 2 neutralizing responses targeting basic residues in strand C of the V2 region, which forms the core epitope for V2-apex bnAbs, were observed. Neutralizing monoclonal antibodies (mAbs) derived from these animals display features promising for subsequent broadening of the response.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Neutralizantes/biosíntesis , Antígenos Virales/inmunología , Anticuerpos Anti-VIH/biosíntesis , Infecciones por VIH/prevención & control , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/biosíntesis , Vacunas contra el SIDA/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Antígenos Virales/química , Antígenos Virales/genética , Sitios de Unión , Epítopos/química , Epítopos/inmunología , Femenino , Anticuerpos Anti-VIH/química , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , Humanos , Inmunización , Pruebas de Neutralización , Polisacáridos/química , Polisacáridos/inmunología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Conejos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Apex broadly neutralizing HIV antibodies (bnAbs) recognize glycans and protein surface close to the 3-fold axis of the envelope (Env) trimer and are among the most potent and broad Abs described. The evolution of apex bnAbs from one donor (CAP256) has been studied in detail and many Abs at different stages of maturation have been described. Using diverse engineering tools, we investigated the involvement of glycan recognition in the development of the CAP256.VRC26 Ab lineage. We found that sialic acid-bearing glycans were recognized by germline-encoded and somatically mutated residues on the Ab heavy chain. This recognition provided an "anchor" for the Abs as the core protein epitope varies, prevented complete neutralization escape, and eventually led to broadening of the response. These findings illustrate how glycan-specific maturation enables a human Ab to cope with pathogen escape mechanisms and will aid in optimization of immunization strategies to induce V2 apex bnAb responses.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/inmunología , Polisacáridos/metabolismo , Secuencia de Aminoácidos , Afinidad de Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , Sitios de Unión , Epítopos/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/clasificación , Anticuerpos Anti-VIH/genética , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/virología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Pruebas de Neutralización , Fragmentos de Péptidos/inmunología , Filogenia , Unión Proteica/inmunología , Conformación Proteica , Multimerización de ProteínaRESUMEN
Broadly neutralizing antibodies (bnAbs) to HIV delineate vaccine targets and are prophylactic and therapeutic agents. Some of the most potent bnAbs target a quaternary epitope at the apex of the surface HIV envelope (Env) trimer. Using cryo-electron microscopy, we solved the atomic structure of an apex bnAb, PGT145, in complex with Env. We showed that the long anionic HCDR3 of PGT145 penetrated between glycans at the trimer 3-fold axis, to contact peptide residues from all three Env protomers, and thus explains its highly trimer-specific nature. Somatic hypermutation in the other CDRs of PGT145 were crucially involved in stabilizing the structure of the HCDR3, similar to bovine antibodies, to aid in recognition of a cluster of conserved basic residues hypothesized to facilitate trimer disassembly during viral entry. Overall, the findings exemplify the creative solutions that the human immune system can evolve to recognize a conserved motif buried under a canopy of glycans.
Asunto(s)
Anticuerpos Neutralizantes/química , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Secuencia de Aminoácidos , Aniones/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Células HEK293 , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/metabolismo , VIH-1/inmunología , VIH-1/metabolismo , Humanos , Modelos Moleculares , Polisacáridos/química , Polisacáridos/inmunología , Polisacáridos/metabolismo , Unión Proteica/inmunología , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by â¼90N-linked glycans, which shield the underlying protein from immune surveillance. bNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields >95% sequence coverage for Env, provides semi-quantitative analysis of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of high-mannose/hybrid and complex-type glycans.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Epítopos/química , Glicosilación , Proteína gp120 de Envoltorio del VIH/química , Espectrometría de Masas , Modelos Moleculares , Péptidos/química , Polisacáridos/química , Multimerización de Proteína , Reproducibilidad de los ResultadosRESUMEN
The wizard of OS (resistance): the binding difference of neuraminidase inhibitors (zanamivir versus oseltamivir (OS)) was used to establish an assay to identify the influenza subtypes that are resistant to OS but still sensitive to zanamivir. This assay used a zanamivir-biotin conjugate to determine the OS susceptibility of a wide range of influenza viruses and over 200 clinical isolates.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Oseltamivir/química , Oseltamivir/farmacología , Unión Competitiva , Farmacorresistencia Viral , Humanos , Subtipo H1N2 del Virus de la Influenza A/efectos de los fármacosRESUMEN
A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, has been prepared to construct a glycan array and used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes, especially from the 2009 swine-originated H1N1 and seasonal influenza viruses. It was found that the HAs from the 2009 H1N1 and the seasonal Brisbane strain share similar binding profiles yet different binding affinities toward various α2,6 sialosides. Analysis of the binding profiles of different HA subtypes indicate that a minimum set of 5 oligosaccharides can be used to differentiate influenza H1, H3, H5, H7, and H9 subtypes. In addition, the glycan array was used to profile the binding pattern of different influenza viruses. It was found that most binding patterns of viruses and HA proteins are similar and that glycosylation at Asn27 is essential for receptor binding.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Polisacáridos/metabolismo , Receptores Virales/metabolismo , Secuencia de Carbohidratos , Línea Celular , Glicosilación , Humanos , Unión ProteicaRESUMEN
As influenza viruses have developed resistance towards current drugs, new inhibitors that prevent viral replication through different inhibitory mechanisms are useful. In this study, we developed a screening procedure to search for new antiinfluenza inhibitors from 1,200,000 compounds and identified previously reported as well as new antiinfluenza compounds. Several antiinfluenza compounds were inhibitory to the influenza RNA-dependent RNA polymerase (RdRP), including nucleozin and its analogs. The most potent nucleozin analog, 3061 (FA-2), inhibited the replication of the influenza A/WSN/33 (H1N1) virus in MDCK cells at submicromolar concentrations and protected the lethal H1N1 infection of mice. Influenza variants resistant to 3061 (FA-2) were isolated and shown to have the mutation on nucleoprotein (NP) that is distinct from the recently reported resistant mutation of Y289H [Kao R, et al. (2010) Nat Biotechnol 28:600]. Recombinant influenza carrying the Y52H NP is also resistant to 3061 (FA-2), and NP aggregation induced by 3061 (FA-2) was identified as the most likely cause for inhibition. In addition, we identified another antiinfluenza RdRP inhibitor 367 which targets PB1 protein but not NP. A mutant resistant to 367 has H456P mutation at the PB1 protein and both the recombinant influenza and the RdRP expressing the PB1 H456P mutation have elevated resistance to 367. Our high-throughput screening (HTS) campaign thus resulted in the identification of antiinfluenza compounds targeting RdRP activity.
Asunto(s)
Antivirales/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Animales , Línea Celular , Perros , Farmacorresistencia Viral/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/enzimología , Gripe Humana/prevención & control , Gripe Humana/virología , Ratones , Nucleoproteínas/genética , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacosRESUMEN
Alpha-galactosyl ceramide (α-GalCer) has been known to bind to the CD1d receptor on dendritic cells and activate invariant natural killer T (iNKT) cells, which subsequently secrete T-helper-cell 1 (Th1) and Th2 cytokines, which correlate with anti-infection activity and the prevention of autoimmune diseases, respectively. α-GalCer elicits the secretion of these two cytokines nonselectively, and thus, its effectiveness is limited by the opposing effects of the Th1 and Th2 cytokines. Reported here is the synthesis of a new α-GalCer analog (compound C34), based on the structure of CD1d, with a 4-(4-fluorophenoxy) phenyl undecanoyl modification of the N-acyl moiety of α-GalCer. Using several murine bacterial and viral infection models, we demonstrated that C34 has superior antibacterial and antiviral activities in comparison with those of several other Th1-selective glycolipids and that it is most effective by administering it to mice in a prophylactic manner before or shortly after infection.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/uso terapéutico , Galactosilceramidas/síntesis química , Galactosilceramidas/uso terapéutico , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Sphingomonas/efectos de los fármacos , Animales , Antibacterianos/administración & dosificación , Antibacterianos/química , Femenino , Galactosilceramidas/administración & dosificación , Galactosilceramidas/química , Infecciones por Bacterias Gramnegativas/microbiología , Ratones , Ratones Endogámicos BALB C , Sphingomonas/patogenicidad , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/patogenicidadRESUMEN
New anti-influenza agents of tetravalent zanamivir on a porphyrin scaffold were synthesized. These compounds are 10 to 100 times more potent in inhibiting influenza replications even though they are somewhat less potent in neuraminidase inhibition than the monomeric zanamivir. The enhanced anti-influenza activity is probably attributable to the additional viral inactivation by singlet oxygen due to sensitization of the porphyrin moiety, which is brought in close proximity of virus by the conjugated zanamivir in a manner resembling the "magic bullet" mechanism.
Asunto(s)
Antivirales/síntesis química , Inhibidores Enzimáticos/síntesis química , Neuraminidasa/antagonistas & inhibidores , Orthomyxoviridae/efectos de los fármacos , Porfirinas/síntesis química , Zanamivir/análogos & derivados , Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Orthomyxoviridae/fisiología , Porfirinas/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Influenza infections are initiated by the binding of the influenza hemagglutinin (HA) and the cellular receptor sialic acids. The binding is followed by internalization, endocytosis, and uncoating to release the influenza genome to the cytoplasm. It is conceivable that specific inhibitors that antagonize any one of these events could prevent the replication of influenza infections. The authors made HA pseudotyped retroviral vectors that express luciferase reporter activities upon transduction to several recipient cells. The transduction of the HA-pseudotype virus particles (HApp) was mediated through the specific interactions between an avian HA and the terminal disaccharides of sialic acid (SA) and galactose (Gal) in alpha-2,3 linkage. The HApp-mediated transduction method was used to develop a high-throughput screening assay and to screen for hits from a fermentation extract library. Specific hits that inhibited the HA-mediated but were noninhibitory to the vesicular stomatitis virus-mediated pseudoviral transductions were identified. A few of these hits have anti-influenza activities that prevent the replication of both H1N1 (WSN) and H5N1 (RG14) influenza viruses.
Asunto(s)
Antivirales/farmacología , Vectores Genéticos/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Animales , Línea Celular , Línea Celular Tumoral , Perros , Evaluación Preclínica de Medicamentos , Genes Reporteros , Vectores Genéticos/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Subtipo H5N1 del Virus de la Influenza A/fisiología , Riñón/citología , Luciferasas/metabolismo , Neoplasias Pulmonares/patología , Plásmidos , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Transducción Genética , Transfección , Replicación Viral/efectos de los fármacosRESUMEN
For the treatment of influenza virus infections, neuraminidase inhibitors (NAIs) that prevent the release of virus particles have been effective against most influenza strains. Several neuraminidase (NA) assays are available for the evaluation of NAIs. To understand the NAI functions under physiological conditions, assays mimicking viral particle release should be useful. We have constructed retrovirus-based reporter viruses that are pseudotyped with hemagglutinin (HA) glycoprotein by transfection of producer cells using plasmids expressing retroviral gag-pol, influenza HA, NA, and firefly luciferase genes. Similarly to the life cycle of influenza viruses, the release of pseudotype viruses also requires neuraminidase functions. This requirement was used to develop an assay to evaluate NAI activities by measuring inhibition of pseudotype virus production at different NAI concentrations. The pseudotype virus release assay was used to determine the IC(50) values of Oseltamivir carboxylate, Zanamivir, and the novel phosphonate congeners of Oseltamivir against N1 group neuraminidases and their H274Y Oseltamivir carboxylate-resistant mutants. The deduced IC(50) values obtained using the release assay correlated with those determined using the fluorogenic substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid (MUNANA) and also correlated with the infectivity results.
Asunto(s)
Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Hemaglutininas Virales/metabolismo , Gripe Humana/tratamiento farmacológico , Neuraminidasa/antagonistas & inhibidores , Orthomyxoviridae/efectos de los fármacos , Animales , Línea Celular , Embrión de Pollo , Evaluación Preclínica de Medicamentos , Genes Reporteros/efectos de los fármacos , Ingeniería Genética , Hemaglutininas Virales/genética , Humanos , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/metabolismo , Neuraminidasa/metabolismo , Orthomyxoviridae/enzimología , Orthomyxoviridae/fisiología , Oseltamivir/farmacología , Esparcimiento de Virus/efectos de los fármacos , Zanamivir/farmacologíaAsunto(s)
Antivirales/síntesis química , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Oseltamivir/análogos & derivados , Oseltamivir/síntesis química , Animales , Antivirales/química , Antivirales/farmacología , Línea Celular , Perros , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H5N1 del Virus de la Influenza A/enzimología , Cinética , Modelos Moleculares , Neuraminidasa/antagonistas & inhibidores , Organofosfonatos/síntesis química , Organofosfonatos/química , Organofosfonatos/farmacología , Oseltamivir/química , Oseltamivir/farmacologíaRESUMEN
Dengue viruses (DENV) are herein demonstrated for the first time as being able to infect and replicate in human primary lung epithelium and various lung cancer cell lines. The detection of dengue virus particles and viral negative strand RNA synthesis in the cell, in conjunction with the release of viral progenies in culture supernatants, support the notion that lung cells are susceptible to dengue virus infection. The replication efficiency of DENV in lung cancer cells from high to low is: DEN-2 (dengue virus type-2), DEN-3, DEN-4 and DEN-1. Moreover, the susceptibility of the six lung cancer cell lines to DEN-2 infection is: SW1573>A549>H1435; H23; H520; Bes2B. DEN-2 infection significantly increased the expression levels of IL-6 and RANTES in four of the six lung cancer cell lines, which is consistent with the high expression levels of these molecules in DHF/DSS patients. IL-6 expression induced by DEN-2 infection was NF-kappaB dependent. In summary, our results indicate that lung epithelial cell is a possible target of dengue viruses and IL-6 and RANTES may play pivotal roles in lung related immuno-pathogenesis.