RESUMEN
Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid-enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA-approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline-treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Animales , Fiebre Hemorrágica Ebola/metabolismo , Ebolavirus/fisiología , Fosfatidilserinas/metabolismo , Fendilina/metabolismo , Proteínas de la Matriz Viral/metabolismo , Ensamble de Virus , Análisis por Conglomerados , Mamíferos/metabolismoRESUMEN
Biosafety, biosecurity, logistical, political, and technical considerations can delay or prevent the wide dissemination of source material containing viable virus from the geographic origin of an outbreak to laboratories involved in developing medical countermeasures (MCMs). However, once virus genome sequence information is available from clinical samples, reverse-genetics systems can be used to generate virus stocks de novo to initiate MCM development. In this study, we developed a reverse-genetics system for natural isolates of Ebola virus (EBOV) variants Makona, Tumba, and Ituri, which have been challenging to obtain. These systems were generated starting solely with in silico genome sequence information and have been used successfully to produce recombinant stocks of each of the viruses for use in MCM testing. The antiviral activity of MCMs targeting viral entry varied depending on the recombinant virus isolate used. Collectively, selecting and synthetically engineering emerging EBOV variants and demonstrating their efficacy against available MCMs will be crucial for answering pressing public health and biosecurity concerns during Ebola disease (EBOD) outbreaks.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/genética , Genética Inversa/métodos , Línea Celular , Brotes de Enfermedades , Ebolavirus/inmunología , Ebolavirus/patogenicidad , Genoma Viral/genética , Genotipo , Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/virología , Humanos , Contramedidas Médicas , Fenotipo , FilogeniaRESUMEN
The severe death toll caused by the recent outbreak of Ebola virus disease reinforces the importance of developing ebolavirus prevention and treatment strategies. Here, we have explored the immunogenicity of a novel immunization regimen priming with vesicular stomatitis virus particles bearing Sudan Ebola virus (SUDV) glycoprotein (GP) that consists of GP1 & GP2 subunits and boosting with soluble SUDV GP in macaques, which developed robust neutralizing antibody (nAb) responses following immunizations. Moreover, EB46, a protective nAb isolated from one of the immune macaques, is found to target the GP1/GP2 interface, with GP-binding mode and neutralization mechanism similar to a number of ebolavirus nAbs from human and mouse, indicating that the ebolavirus GP1/GP2 interface is a common immunological target in different species. Importantly, selected immune macaque polyclonal sera showed nAb specificity similar to EB46 at substantial titers, suggesting that the GP1/GP2 interface region is a viable target for ebolavirus vaccine.Importance: The elicitation of sustained neutralizing antibody (nAb) responses against diverse ebolavirus strains remains as a high priority for the vaccine field. The most clinically advanced rVSV-ZEBOV vaccine could elicit moderate nAb responses against only one ebolavirus strain, EBOV, among the five ebolavirus strains, which last less than 6 months. Boost immunization strategies are desirable to effectively recall the rVSV vector-primed nAb responses to prevent infections in prospective epidemics, while an in-depth understanding of the specificity of immunization-elicited nAb responses is essential for improving vaccine performance. Here, using non-human primate animal model, we demonstrated that booster immunization with a stabilized trimeric soluble form of recombinant glycoprotein derived from the ebolavirus Sudan strain following the priming rVSV vector immunization led to robust nAb responses that substantially map to the subunit interface of ebolavirus glycoprotein, a common B cell repertoire target of multiple species including primates and rodents.
RESUMEN
Global health is threatened by emerging viruses, many of which lack approved therapies and effective vaccines, including dengue, Ebola, and Venezuelan equine encephalitis. We previously reported that AAK1 and GAK, two of the four members of the understudied Numb-associated kinases (NAK) family, control intracellular trafficking of RNA viruses. Nevertheless, the role of BIKE and STK16 in viral infection remained unknown. Here, we reveal a requirement for BIKE, but not STK-16, in dengue virus (DENV) infection. BIKE mediates both early (postinternalization) and late (assembly/egress) stages in the DENV life cycle, and this effect is mediated in part by phosphorylation of a threonine 156 (T156) residue in the µ subunit of the adaptor protein (AP) 2 complex. Pharmacological compounds with potent anti-BIKE activity, including the investigational anticancer drug 5Z-7-oxozeaenol and more selective inhibitors, suppress DENV infection both in vitro and ex vivo. BIKE overexpression reverses the antiviral activity, validating that the mechanism of antiviral action is, at least in part, mediated by BIKE. Lastly, 5Z-7-oxozeaenol exhibits antiviral activity against viruses from three unrelated RNA viral families with a high genetic barrier to resistance. These findings reveal regulation of poorly understood stages of the DENV life cycle via BIKE signaling and establish a proof-of-principle that pharmacological inhibition of BIKE can be potentially used as a broad-spectrum strategy against acute emerging viral infections.
Asunto(s)
Virus del Dengue/fisiología , Dengue/virología , Lactonas/farmacología , Proteínas Serina-Treonina Quinasas/fisiología , Resorcinoles/farmacología , Factores de Transcripción/fisiología , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Animales , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Dengue/tratamiento farmacológico , Virus del Dengue/efectos de los fármacos , Reposicionamiento de Medicamentos , Interacciones Microbiota-Huesped , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , ARN Viral , Proteínas Recombinantes , Transducción de Señal , Células Vero , Internalización del Virus , Replicación ViralRESUMEN
There are currently no approved drugs for the treatment of emerging viral infections, such as dengue and Ebola. Adaptor-associated kinase 1 (AAK1) is a cellular serine-threonine protein kinase that functions as a key regulator of the clathrin-associated host adaptor proteins and regulates the intracellular trafficking of multiple unrelated RNA viruses. Moreover, AAK1 is overexpressed specifically in dengue virus-infected but not bystander cells. Because AAK1 is a promising antiviral drug target, we have embarked on an optimization campaign of a previously identified 7-azaindole analogue, yielding novel pyrrolo[2,3- b]pyridines with high AAK1 affinity. The optimized compounds demonstrate improved activity against dengue virus both in vitro and in human primary dendritic cells and the unrelated Ebola virus. These findings demonstrate that targeting cellular AAK1 may represent a promising broad-spectrum antiviral strategy.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/síntesis química , Piridinas/farmacología , Antivirales/química , Antivirales/metabolismo , Línea Celular , Técnicas de Química Sintética , Humanos , Simulación del Acoplamiento Molecular , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/química , Piridinas/metabolismo , Relación Estructura-ActividadRESUMEN
The 2013-2016 Ebola virus (EBOV) disease epidemic demonstrated the grave consequences of filovirus epidemics in the absence of effective therapeutics. Besides EBOV, two additional ebolaviruses, Sudan (SUDV) and Bundibugyo (BDBV) viruses, as well as multiple variants of Marburg virus (MARV), have also caused high fatality epidemics. Current experimental EBOV monoclonal antibodies (mAbs) are ineffective against SUDV, BDBV, or MARV. Here, we report that a cocktail of two broadly neutralizing ebolavirus mAbs, FVM04 and CA45, protects nonhuman primates (NHPs) against EBOV and SUDV infection when delivered four days post infection. This cocktail when supplemented by the anti-MARV mAb MR191 exhibited 100% efficacy in MARV-infected NHPs. These findings provide a solid foundation for clinical development of broadly protective immunotherapeutics for use in future filovirus epidemics.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Ebolavirus/inmunología , Infecciones por Filoviridae/inmunología , Marburgvirus/inmunología , Enfermedades de los Primates/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Ebolavirus/clasificación , Ebolavirus/efectos de los fármacos , Ebolavirus/fisiología , Infecciones por Filoviridae/terapia , Infecciones por Filoviridae/virología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Inmunoterapia/métodos , Marburgvirus/efectos de los fármacos , Marburgvirus/fisiología , Enfermedades de los Primates/terapia , Enfermedades de los Primates/virología , Primates , Resultado del TratamientoRESUMEN
Sexual transmission of filoviruses was first reported in 1968 after an outbreak of Marburg virus (MARV) disease and recently caused flare-ups of Ebola virus disease in the 2013-2016 outbreak. How filoviruses establish testicular persistence and are shed in semen remain unknown. We discovered that persistent MARV infection of seminiferous tubules, an immune-privileged site that harbors sperm production, is a relatively common event in crab-eating macaques that survived infection after antiviral treatment. Persistence triggers severe testicular damage, including spermatogenic cell depletion and inflammatory cell invasion. MARV mainly persists in Sertoli cells, leading to breakdown of the blood-testis barrier formed by inter-Sertoli cell tight junctions. This disruption is accompanied by local infiltration of immunosuppressive CD4+Foxp3+ regulatory T cells. Our study elucidates cellular events associated with testicular persistence that may promote sexual transmission of filoviruses and suggests that targeting immunosuppression may be warranted to clear filovirus persistence in damaged immune-privileged sites.
Asunto(s)
Enfermedad del Virus de Marburg/virología , Marburgvirus/fisiología , Enfermedades de los Primates/virología , Testículo/virología , Animales , Macaca , Masculino , Enfermedad del Virus de Marburg/inmunología , Enfermedad del Virus de Marburg/metabolismo , Enfermedades de los Primates/inmunología , Enfermedades de los Primates/metabolismo , Células de Sertoli/metabolismo , Células de Sertoli/virología , Sobrevivientes , Linfocitos T Reguladores/inmunología , Uniones Estrechas/metabolismo , Uniones Estrechas/virologíaRESUMEN
Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Ebolavirus/inmunología , Epítopos/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Glicoproteínas de Membrana/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Femenino , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/virología , Inmunización , Ratones , Ratones Endogámicos BALB C , Resultado del TratamientoRESUMEN
The recent Ebola virus (EBOV) epidemic highlighted the need for effective vaccines and therapeutics to limit and prevent outbreaks. Host antibodies against EBOV are critical for controlling disease, and recombinant monoclonal antibodies (mAbs) can protect from infection. However, antibodies mediate an array of antiviral functions including neutralization as well as engagement of Fc-domain receptors on immune cells, resulting in phagocytosis or NK cell-mediated killing of infected cells. Thus, to understand the antibody features mediating EBOV protection, we examined specific Fc features associated with protection using a library of EBOV-specific mAbs. Neutralization was strongly associated with therapeutic protection against EBOV. However, several neutralizing mAbs failed to protect, while several non-neutralizing or weakly neutralizing mAbs could protect. Antibody-mediated effector functions, including phagocytosis and NK cell activation, were associated with protection, particularly for antibodies with moderate neutralizing activity. This framework identifies functional correlates that can inform therapeutic and vaccine design strategies against EBOV and other pathogens.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Fragmentos Fc de Inmunoglobulinas/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Femenino , Fiebre Hemorrágica Ebola/inmunología , Humanos , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Células RAW 264.7RESUMEN
There is an urgent need for strategies to combat dengue and other emerging viral infections. We reported that cyclin G-associated kinase (GAK), a cellular regulator of the clathrin-associated host adaptor proteins AP-1 and AP-2, regulates intracellular trafficking of multiple unrelated RNA viruses during early and late stages of the viral lifecycle. We also reported the discovery of potent, selective GAK inhibitors based on an isothiazolo[4,3- b]pyridine scaffold, albeit with moderate antiviral activity. Here, we describe our efforts leading to the discovery of novel isothiazolo[4,3- b]pyridines that maintain high GAK affinity and selectivity. These compounds demonstrate improved in vitro activity against dengue virus, including in human primary dendritic cells, and efficacy against the unrelated Ebola and chikungunya viruses. Moreover, inhibition of GAK activity was validated as an important mechanism of antiviral action of these compounds. These findings demonstrate the potential utility of a GAK-targeted broad-spectrum approach for combating currently untreatable emerging viral infections.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/química , Piridinas/farmacología , Tiazoles/química , Antivirales/química , Antivirales/farmacología , Línea Celular , Humanos , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
Sudan virus (SUDV) and Ebola viruses (EBOV) are both members of the Ebolavirus genus and have been sources of epidemics and outbreaks for several decades. We present here the generation and characterization of cross-reactive antibodies to both SUDV and EBOV, which were produced in a cell-free system and protective against SUDV in mice. A non-human primate, cynomolgus macaque, was immunized with viral-replicon particles expressing the glycoprotein of SUDV-Boniface (8A). Two separate antibody fragment phage display libraries were constructed after four immunogen injections. Both libraries were screened first against the SUDV and a second library was cross-selected against EBOV-Kikwit. Sequencing of 288 selected clones from the two distinct libraries identified 58 clones with distinct VH and VL sequences. Many of these clones were cross-reactive to EBOV and SUDV and able to neutralize SUDV. Three of these recombinant antibodies (X10B1, X10F3, and X10H2) were produced in the scFv-Fc format utilizing a cell-free production system. Mice that were challenged with SUDV-Boniface receiving 100µg of the X10B1/X10H2 scFv-Fc combination 6 and 48-h post-exposure demonstrated partial protection individually and complete protection as a combination. The data herein suggests these antibodies may be promising candidates for further therapeutic development.
Asunto(s)
Anticuerpos Antivirales/farmacología , Ebolavirus , Fiebre Hemorrágica Ebola/terapia , Glicoproteínas de Membrana/inmunología , Profilaxis Posexposición , Vacunas de Partículas Similares a Virus/inmunología , Proteínas de la Matriz Viral/inmunología , Animales , Anticuerpos Neutralizantes/farmacología , Técnicas de Visualización de Superficie Celular , Reacciones Cruzadas , Femenino , Macaca , Masculino , Ratones , Ratones Noqueados , Anticuerpos de Cadena Única/farmacología , VacunaciónRESUMEN
While neutralizing antibodies are highly effective against ebolavirus infections, current experimental ebolavirus vaccines primarily elicit species-specific antibody responses. Here, we describe an immunization-elicited macaque antibody (CA45) that clamps the internal fusion loop with the N terminus of the ebolavirus glycoproteins (GPs) and potently neutralizes Ebola, Sudan, Bundibugyo, and Reston viruses. CA45, alone or in combination with an antibody that blocks receptor binding, provided full protection against all pathogenic ebolaviruses in mice, guinea pigs, and ferrets. Analysis of memory B cells from the immunized macaque suggests that elicitation of broadly neutralizing antibodies (bNAbs) for ebolaviruses is possible but difficult, potentially due to the rarity of bNAb clones and their precursors. Unexpectedly, germline-reverted CA45, while exhibiting negligible binding to full-length GP, bound a proteolytically remodeled GP with picomolar affinity, suggesting that engineered ebolavirus vaccines could trigger rare bNAb precursors more robustly. These findings have important implications for developing pan-ebolavirus vaccine and immunotherapeutic cocktails.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Vacunas contra el Virus del Ébola/inmunología , Fiebre Hemorrágica Ebola/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Regiones Determinantes de Complementariedad , Reacciones Cruzadas , Ebolavirus/inmunología , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Femenino , Hurones , Cobayas , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos MolecularesRESUMEN
Drug combinations are synergistic when their combined efficacy exceeds the sum of the individual actions, but they rarely include ineffective drugs that become effective only in combination. We identified several "enabling pairs" of neutralizing and non-neutralizing anti-ebolavirus monoclonal antibodies, whose combination exhibited new functional profiles, including transforming a non-neutralizing antibody to a neutralizer. Sub-neutralizing concentrations of antibodies 2G4 or m8C4 enabled non-neutralizing antibody FVM09 (IC50 >1 µM) to exhibit potent neutralization (IC50 1-10 nM). While FVM09 or m8C4 alone failed to protect Ebola-virus-infected mice, a combination of the two antibodies provided 100% protection. Furthermore, non-neutralizers FVM09 and FVM02 exponentially enhanced the potency of two neutralizing antibodies against both Ebola and Sudan viruses. We identified a hotspot for the binding of these enabling antibody pairs near the interface of the glycan cap and GP2. Enabling cooperativity may be an underappreciated phenomenon for viruses, with implications for the design and development of immunotherapeutics and vaccines.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Sinergismo Farmacológico , Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/terapia , Fiebre Hemorrágica Ebola/virología , Humanos , RatonesRESUMEN
Marburg virus (MARV) and Ebola virus (EBOV) have been a source of epidemics and outbreaks for several decades. We present here the generation and characterization of the first protective antibodies specific for wild-type MARV. Non-human primates (NHP), cynomolgus macaques, were immunized with viral-replicon particles expressing the glycoproteins (GP) of MARV (Ci67 isolate). An antibody fragment (single-chain variable fragment, scFv) phage display library was built after four immunogen injections, and screened against the GP1-649 of MARV. Sequencing of 192 selected clones identified 18 clones with distinct VH and VL sequences. Four of these recombinant antibodies (R4A1, R4B11, R4G2, and R3F6) were produced in the scFv-Fc format for in vivo studies. Mice that were challenged with wild-type Marburg virus (Ci67 isolate) receiving 100 µg of scFv-Fc on days -1, 1 and 3 demonstrated protective efficacies ranging from 75-100%. The amino-acid sequences of the scFv-Fcs are similar to those of their human germline counterparts, sharing an identity ranging between 68 and 100% to human germline immunoglobulin. These results demonstrate for the first time that recombinant antibodies offer protection against wild-type MARV, and suggest they may be promising candidates for further therapeutic development especially due to their human homology.
Asunto(s)
Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Marburgvirus/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Humanos , Macaca fascicularisRESUMEN
Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.
Asunto(s)
Antineoplásicos/farmacología , Antivirales/farmacología , Clorhidrato de Erlotinib/farmacología , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Complejo 1 de Proteína Adaptadora/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Línea Celular Tumoral , Dengue/prevención & control , Dengue/virología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/metabolismo , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Ebolavirus/efectos de los fármacos , Ebolavirus/metabolismo , Femenino , Fiebre Hemorrágica Ebola/prevención & control , Fiebre Hemorrágica Ebola/virología , Hepacivirus/efectos de los fármacos , Hepacivirus/metabolismo , Hepatitis C/prevención & control , Hepatitis C/virología , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Sunitinib , Internalización del Virus/efectos de los fármacosRESUMEN
Previous efforts to identify cross-neutralizing antibodies to the receptor-binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS; cross-neutralizes Ebola (EBOV), Sudan (SUDV), and, to a lesser extent, Bundibugyo viruses; and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™ is remarkably effective against EBOV (Zaire) but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross-protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Ebolavirus/fisiología , Epítopos/inmunología , Glicoproteínas/metabolismo , Fiebre Hemorrágica Ebola/inmunología , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/ultraestructura , Anticuerpos Neutralizantes , Anticuerpos Antivirales/química , Sitios de Unión , Modelos Animales de Enfermedad , Femenino , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/ultraestructura , Cobayas , Células HEK293 , Humanos , Cinética , Ratones Endogámicos BALB C , Modelos Moleculares , Mutación/genética , Coloración Negativa , Pruebas de Neutralización , Resultado del TratamientoRESUMEN
Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities.
Asunto(s)
Animales Modificados Genéticamente , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/uso terapéutico , Bovinos , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunización Pasiva , Inmunoglobulina G/sangre , Inmunoglobulina G/uso terapéutico , Ratones Endogámicos BALB C , Pruebas de Neutralización , Resonancia por Plasmón de Superficie , Resultado del TratamientoRESUMEN
DNA vaccination of transchromosomal bovines (TcBs) with DNA vaccines expressing the codon-optimized (co) glycoprotein (GP) genes of Ebola virus (EBOV) and Sudan virus (SUDV) produce fully human polyclonal antibodies (pAbs) that recognize both viruses and demonstrate robust neutralizing activity. Each TcB was vaccinated by intramuscular electroporation (IM-EP) a total of four times and at each administration received 10 mg of the EBOV-GPco DNA vaccine and 10 mg of the SUDV-GPco DNA vaccine at two sites on the left and right sides, respectively. After two vaccinations, robust antibody responses (titers > 1000) were detected by ELISA against whole irradiated EBOV or SUDV and recombinant EBOV-GP or SUDV-GP (rGP) antigens, with higher titers observed for the rGP antigens. Strong, virus neutralizing antibody responses (titers >1000) were detected after three vaccinations when measured by vesicular stomatitis virus-based pseudovirion neutralization assay (PsVNA). Maximal neutralizing antibody responses were identified by traditional plaque reduction neutralization tests (PRNT) after four vaccinations. Neutralizing activity of human immunoglobulins (IgG) purified from TcB plasma collected after three vaccinations and injected intraperitoneally (IP) into mice at a 100 mg/kg dose was detected in the serum by PsVNA up to 14 days after administration. Passive transfer by IP injection of the purified IgG (100 mg/kg) to groups of BALB/c mice one day after IP challenge with mouse adapted (ma) EBOV resulted in 80% protection while all mice treated with non-specific pAbs succumbed. Similarly, interferon receptor 1 knockout (IFNAR(-/-)) mice receiving the purified IgG (100 mg/kg) by IP injection one day after IP challenge with wild type SUDV resulted in 89% survival. These results are the first to demonstrate that filovirus GP DNA vaccines administered to TcBs by IM-EP can elicit neutralizing antibodies that provide post-exposure protection. Additionally, these data describe production of fully human IgG in a large animal system, a system which is capable of producing large quantities of a clinical grade therapeutic product.
Asunto(s)
Anticuerpos Antivirales/metabolismo , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Profilaxis Posexposición , Vacunas de ADN/inmunología , Animales , Animales Modificados Genéticamente , Anticuerpos Neutralizantes/inmunología , Bovinos/genética , Bovinos/inmunología , Cromosomas Artificiales Humanos/genética , República Democrática del Congo , Vacunas contra el Virus del Ébola/inmunología , Femenino , Fiebre Hemorrágica Ebola/virología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Profilaxis Posexposición/métodos , Receptor de Interferón alfa y beta/genética , Sudán , Vacunación/métodosRESUMEN
A major obstacle in ebolavirus research is the lack of a small-animal model for Sudan virus (SUDV), as well as other wild-type (WT) ebolaviruses. Here, we expand on research by Bray and by Lever et al suggesting that WT ebolaviruses are pathogenic in mice deficient for the type 1 interferon (IFN) α/ß receptor (IFNα/ßR-/-). We examined the disease course of several WT ebolaviruses: Boneface (SUDV/Bon) and Gulu variants of SUDV, Ebola virus (EBOV), Bundibugyo virus (BDBV), Taï Forest virus, and Reston virus (RESTV). We determined that exposure to WT SUDV or EBOV results in reproducible signs of disease in IFNα/ßR-/- mice, as measured by weight loss and partial lethality. Vaccination with the SUDV or EBOV glycoprotein (GP)-expressing Venezuelan equine encephalitis viral replicon particle vaccine protected these mice from SUDV/Bon and EBOV challenge, respectively. Treatment with SUDV- or EBOV-specific anti-GP antibodies protected mice from challenge when delivered 1-3 days after infection. Serial sampling experiments revealed evidence of disseminated intravascular coagulation in the livers of mice infected with the Boneface variant of SUDV, EBOV, and BDBV. Taken together, these data solidify the IFNα/ßR-/- mouse as an important and useful model for the study of WT EBOV disease.
Asunto(s)
Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/virología , Receptor de Interferón alfa y beta/deficiencia , Virulencia/fisiología , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Chlorocebus aethiops , Modelos Animales de Enfermedad , Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/metabolismo , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Ratones , Ratones Endogámicos C57BL , Replicón/inmunología , Vacunación/métodos , Células Vero/virología , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Virulencia/inmunologíaRESUMEN
Ebola viruses remain a substantial threat to both civilian and military populations as bioweapons, during sporadic outbreaks, and from the possibility of accidental importation from endemic regions by infected individuals. Currently, no approved therapeutics exist to treat or prevent infection by Ebola viruses. Therefore, we performed an in vitro screen of Food and Drug Administration (FDA)- and ex-US-approved drugs and selected molecular probes to identify drugs with antiviral activity against the type species Zaire ebolavirus (EBOV). From this screen, we identified a set of selective estrogen receptor modulators (SERMs), including clomiphene and toremifene, which act as potent inhibitors of EBOV infection. Anti-EBOV activity was confirmed for both of these SERMs in an in vivo mouse infection model. This anti-EBOV activity occurred even in the absence of detectable estrogen receptor expression, and both SERMs inhibited virus entry after internalization, suggesting that clomiphene and toremifene are not working through classical pathways associated with the estrogen receptor. Instead, the response appeared to be an off-target effect where the compounds interfere with a step late in viral entry and likely affect the triggering of fusion. These data support the screening of readily available approved drugs to identify therapeutics for the Ebola viruses and other infectious diseases. The SERM compounds described in this report are an immediately actionable class of approved drugs that can be repurposed for treatment of filovirus infections.