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1.
Mil Med ; 2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33428750

RESUMO

INTRODUCTION: Dengue fever, caused by any of the four dengue viruses (DENV1-4), is endemic in more than 100 countries around the world. Each year, up to 400 million people get infected with dengue virus. It is one of the most important arthropod-borne viral diseases. Dengue's global presence poses a medical threat to deploying military personnel and their dependents. An accurate diagnosis followed by attentive supportive care can improve outcomes in patients with severe dengue disease. Dengue diagnostic tests based on PCR and ELISA platforms have been developed and cleared by the U.S. FDA. However, these diagnostic assays are laborious and usually require highly trained personnel and specialized equipment, which presents a significant challenge when conducting operations in austere and resource-constrained areas. InBios International, Inc. (Seattle, WA) has developed two rapid and instrument-free immunochromatographic test prototype devices (multiplex and traditional formats) for dengue diagnosis. MATERIALS AND METHODS: To determine the performance of the InBios immunochromatographic tests, 183 clinical samples were tested on both prototype devices. Both assays were performed without any instruments and the results were read in 20 minutes. RESULTS: The traditional format had better overall performance (sensitivity: 97.4%; specificity: 90%) than the multiplex format (sensitivity: 86.9%; specificity: 63.3%). The traditional format was superior in serotype-specific detection with 100% overall sensitivity for DENV1, DENV3, and DENV4 and 93.3% sensitivity for DENV2 compared to the multiplex format (91.7%, 78.3%, 83.3%, and 96.3% for DENV1, 2, 3, and 4, respectively). The traditional format was easier to read than the multiplex format. The multiplex format was simpler and faster to set up than the traditional format. CONCLUSIONS: The InBios traditional format had a better overall performance and readability profile than the multiplex format, while the multiplex format was easier to set up. Both formats were highly sensitive and specific, were easy to perform, and did not require sophisticated equipment. They are ideal for use in resource-limited settings where dengue is endemic. Based on our overall assessment, the traditional format should be considered for further development and used in the upcoming multicenter clinical trial toward FDA clearance.

2.
J Virol ; 90(1): 279-91, 2016 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-26468532

RESUMO

UNLABELLED: Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE: Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Filoviridae/imunologia , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/uso terapêutico , Reações Cruzadas , Modelos Animais de Doenças , Feminino , Imunização Passiva , Macaca , Camundongos Endogâmicos BALB C , Análise de Sobrevida , Resultado do Tratamento
3.
J Virol ; 90(1): 266-78, 2016 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-26468533

RESUMO

UNLABELLED: The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. IMPORTANCE: Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective immunotherapeutics are urgently needed. Here, we describe monoclonal antibodies with cross-reactivity to several filoviruses, including the first report of a cross-neutralizing antibody that exhibits protection against Ebola virus and Sudan virus in mice. Our results further describe a novel combination of antibodies with enhanced protective efficacy. These results form a basis for further development of effective immunotherapeutics against filoviruses for human use. Understanding the cross-protective epitopes are also important for rational design of pan-ebolavirus and pan-filovirus vaccines.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Filoviridae/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Imunização Passiva , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/administração & dosagem , Proteção Cruzada , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Camundongos Endogâmicos BALB C , Resultado do Tratamento
4.
J Virol ; 89(19): 9932-8, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26202243

RESUMO

UNLABELLED: Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. IMPORTANCE: Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. Our results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.


Assuntos
Ebolavirus/efeitos dos fármacos , Ebolavirus/fisiologia , Marburgvirus/efeitos dos fármacos , Marburgvirus/fisiologia , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/farmacologia , Benzotropina/farmacologia , Linhagem Celular , Chlorocebus aethiops , Ciproeptadina/farmacologia , Ebolavirus/patogenicidade , Células HEK293 , Doença pelo Vírus Ebola/tratamento farmacológico , Heparina/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Macrolídeos/farmacologia , Doença do Vírus de Marburg/tratamento farmacológico , Marburgvirus/patogenicidade , Receptores Acoplados a Proteínas-G/fisiologia , Bibliotecas de Moléculas Pequenas , Células Vero , Zidovudina/farmacologia
5.
Sci Transl Med ; 7(290): 290ra89, 2015 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-26041706

RESUMO

Currently, no approved therapeutics exist to treat or prevent infections induced by Ebola viruses, and recent events have demonstrated an urgent need for rapid discovery of new treatments. Repurposing approved drugs for emerging infections remains a critical resource for potential antiviral therapies. We tested ~2600 approved drugs and molecular probes in an in vitro infection assay using the type species, Zaire ebolavirus. Selective antiviral activity was found for 80 U.S. Food and Drug Administration-approved drugs spanning multiple mechanistic classes, including selective estrogen receptor modulators, antihistamines, calcium channel blockers, and antidepressants. Results using an in vivo murine Ebola virus infection model confirmed the protective ability of several drugs, such as bepridil and sertraline. Viral entry assays indicated that most of these antiviral drugs block a late stage of viral entry. By nature of their approved status, these drugs have the potential to be rapidly advanced to clinical settings and used as therapeutic countermeasures for Ebola virus infections.


Assuntos
Antivirais/uso terapêutico , Aprovação de Drogas , Doença pelo Vírus Ebola/terapia , Sondas Moleculares , Animais , Bepridil/farmacologia , Ebolavirus/efeitos dos fármacos , Humanos , Camundongos , Sertralina/farmacologia
6.
J Virol ; 89(10): 5441-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25741008

RESUMO

UNLABELLED: Filoviruses, including both Ebola virus (EBOV) and Marburg virus (MARV), can infect humans and other animals, causing hemorrhagic fever with a high mortality rate. Entry of these viruses into the host is mediated by a single filoviral glycoprotein (GP). GP is composed of two subunits: GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. Although numerous host factors have been implicated in the entry process, the initial attachment receptor(s) has not been well defined. In this report, we demonstrate that exostosin 1 (EXT1), which is involved in biosynthesis of heparan sulfate (HS), plays a role in filovirus entry. Expression knockdown of EXT1 by small interfering RNAs (siRNAs) impairs GP-mediated pseudoviral entry and that of infectious EBOV and MARV in tissue cultured cells. Furthermore, HS, heparin, and other related glycosaminoglycans (GAGs), to different extents, can bind to and block GP-mediated viral entry and that of infectious filoviruses. These results strongly suggest that HS and other related GAGs are attachment receptors that are utilized by filoviruses for entry and infection. These GAGs may have therapeutic potential in treating EBOV- and MARV-infected patients. IMPORTANCE: Infection by Ebola virus and Marburg virus can cause severe illness in humans, with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The ongoing 2014 outbreak in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we provide several pieces of evidence that demonstrate that heparan sulfate and other closely related glycosaminoglycans are the molecules that are used by filoviruses for initial attachment. Furthermore, we demonstrate that these glycosaminoglycans can block entry of and infection by filoviruses. Thus, this work provides mechanistic insights on the early step of filoviral infection and suggests a possible therapeutic option for diseases caused by filovirus infection.


Assuntos
Filoviridae/fisiologia , Glicosaminoglicanos/fisiologia , N-Acetilglucosaminiltransferases/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Ebolavirus/patogenicidade , Ebolavirus/fisiologia , Filoviridae/patogenicidade , Infecções por Filoviridae/etiologia , Infecções por Filoviridae/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Heparina/fisiologia , Heparitina Sulfato/biossíntese , Heparitina Sulfato/deficiência , Interações Hospedeiro-Patógeno , Humanos , Marburgvirus/patogenicidade , Marburgvirus/fisiologia , Camundongos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , Receptores Virais/fisiologia , Proteínas Virais/fisiologia , Virulência
7.
Antiviral Res ; 112: 1-7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25265598

RESUMO

The cyanobacterial lectin scytovirin (SVN) binds with high affinity to mannose-rich oligosaccharides on the envelope glycoprotein (GP) of a number of viruses, blocking entry into target cells. In this study, we assessed the ability of SVN to bind to the envelope GP of Zaire Ebola virus (ZEBOV) and inhibit its replication. SVN interacted specifically with the protein's mucin-rich domain. In cell culture, it inhibited ZEBOV replication with a 50% virus-inhibitory concentration (EC50) of 50 nM, and was also active against the Angola strain of the related Marburg virus (MARV), with a similar EC50. Injected subcutaneously in mice, SVN reached a peak plasma level of 100 nm in 45 min, but was cleared within 4h. When ZEBOV-infected mice were given 30 mg/kg/day of SVN by subcutaneous injection every 6h, beginning the day before virus challenge, 9 of 10 animals survived the infection, while all infected, untreated mice died. When treatment was begun one hour or one day after challenge, 70-90% of mice survived. Quantitation of infectious virus and viral RNA in samples of serum, liver and spleen collected on days 2 and 5 postinfection showed a trend toward lower titers in treated than control mice, with a significant decrease in liver titers on day 2. Our findings provide further evidence of the potential of natural lectins as therapeutic agents for viral infections.


Assuntos
Antivirais/uso terapêutico , Proteínas de Bactérias/uso terapêutico , Proteínas de Transporte/uso terapêutico , Ebolavirus/efeitos dos fármacos , Lectinas/uso terapêutico , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , Antivirais/metabolismo , Antivirais/farmacologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Modelos Animais de Doenças , Ebolavirus/fisiologia , Glicoproteínas/metabolismo , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Concentração Inibidora 50 , Injeções Subcutâneas , Lectinas/administração & dosagem , Lectinas/metabolismo , Lectinas/farmacologia , Fígado/virologia , Marburgvirus/efeitos dos fármacos , Proteínas de Membrana , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Soro/virologia , Baço/virologia , Análise de Sobrevida , Carga Viral
8.
Antimicrob Agents Chemother ; 58(8): 4885-93, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24841273

RESUMO

Outbreaks of emerging infections present health professionals with the unique challenge of trying to select appropriate pharmacologic treatments in the clinic with little time available for drug testing and development. Typically, clinicians are left with general supportive care and often untested convalescent-phase plasma as available treatment options. Repurposing of approved pharmaceutical drugs for new indications presents an attractive alternative to clinicians, researchers, public health agencies, drug developers, and funding agencies. Given the development times and manufacturing requirements for new products, repurposing of existing drugs is likely the only solution for outbreaks due to emerging viruses. In the studies described here, a library of 290 compounds was screened for antiviral activity against Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Selection of compounds for inclusion in the library was dependent on current or previous FDA approval or advanced clinical development. Some drugs that had a well-defined cellular pathway as target were included. In total, 27 compounds with activity against both MERS-CoV and SARS-CoV were identified. The compounds belong to 13 different classes of pharmaceuticals, including inhibitors of estrogen receptors used for cancer treatment and inhibitors of dopamine receptor used as antipsychotics. The drugs identified in these screens provide new targets for in vivo studies as well as incorporation into ongoing clinical studies.


Assuntos
Antivirais/farmacologia , Reposicionamento de Medicamentos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Vírus da SARS/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antipsicóticos/farmacologia , Chlorocebus aethiops , Aprovação de Drogas , Antagonistas de Estrogênios/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Vírus da SARS/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacos
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