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1.
Microb Pathog ; 132: 243-253, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31075428

RESUMO

Ebola virus (EBOV), a non-segmented single-stranded RNA virus, is often-most transmitted through body fluids like sweat, tears, saliva, and nasal secretions. Till date, there is no licensed vaccine of EBOV is available in the market; however, the world is increasingly vulnerable to this emerging threat. Hence, it is the need of time to develop a vaccine for EBOV to hinder its dissemination. The current study has been designed for identification and characterization of the potential B and T-cell epitopes using the Immuno-informatics tools, and it helped in finding the potent vaccine candidates against EBOV. Prediction, antigenicity and allergenicity testing of predicted B and T cells' epitopes was done as well to identify their potential as a vaccine candidate and to measure their safety level respectively. Among B-cell epitopes "WIPAGIGVTGVIIA" showed a high antigenicity score and it would play an important role in evoking the immune response. In T-cell epitopes, peptides "AIGLAWIPY" and "IRGFPRCRY" presented high antigenicity score, which binds to MHC class-I and MHC class-II alleles respectively. All predicted epitopes were analyzed and compared with already reported peptides carefully. Comparatively, Peptides predicted in the present study showed more immunogenicity score than already reported peptides, used as positive control, and are more immunogenic as compared to them. Peptides reported in the present study do not target only Zaire EBOV (ZEBOV), as in previous studies, but also other species, i.e. Tai Forest EBOV (TAFV), Sudan EBOV (SUDV), Bundibugyo EBOV (BDBV), and Reston EBOV (RESTV) and would bring the promising results as potent vaccine candidates.


Assuntos
Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Glicoproteínas/imunologia , Alelos , Sequência de Aminoácidos , Vacinas contra Ebola/genética , Ebolavirus/genética , Genes MHC Classe I , Genes MHC da Classe II , Glicoproteínas/química , Glicoproteínas/genética , Antígeno HLA-B7 , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Estrutura Secundária de Proteína
2.
Nat Commun ; 10(1): 1788, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996276

RESUMO

Three Ebolavirus genus viruses cause lethal disease and lack targeted therapeutics: Ebola virus, Sudan virus and Bundibugyo virus. Monoclonal antibody (mAb) cocktails against the surface glycoprotein (GP) present a potential therapeutic strategy. Here we report two crystal structures of the antibody BDBV223, alone and complexed with its GP2 stalk epitope, an interesting site for therapeutic/vaccine design due to its high sequence conservation among ebolaviruses. BDBV223, identified in a human survivor of Bundibugyo virus disease, neutralizes both Bundibugyo virus and Ebola virus, but not Sudan virus. Importantly, the structure suggests that BDBV223 binding interferes with both the trimeric bundle assembly of GP and the viral membrane by stabilizing a conformation in which the monomers are separated by GP lifting or bending. Targeted mutagenesis of BDBV223 to enhance SUDV GP recognition indicates that additional determinants of antibody binding likely lie outside the visualized interactions, and perhaps involve quaternary assembly or membrane-interacting regions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , Reações Cruzadas/imunologia , Cristalografia por Raios X , Ebolavirus/imunologia , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Hibridomas , Mutagênese , Sobreviventes , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
3.
Proc Natl Acad Sci U S A ; 116(13): 5886-5891, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30846548

RESUMO

Proteins are widely regarded as insulators, despite reports of electrical conductivity. Here we use measurements of single proteins between electrodes, in their natural aqueous environment to show that the factor controlling measured conductance is the nature of the electrical contact to the protein, and that specific ligands make highly selective electrical contacts. Using six proteins that lack known electrochemical activity, and measuring in a potential region where no ion current flows, we find characteristic peaks in the distributions of measured single-molecule conductances. These peaks depend on the contact chemistry, and hence, on the current path through the protein. In consequence, the measured conductance distribution is sensitive to changes in this path caused by ligand binding, as shown with streptavidin-biotin complexes. Measured conductances are on the order of nanosiemens over distances of many nanometers, orders of magnitude more than could be accounted for by electron tunneling. The current is dominated by contact resistance, so the conductance for a given path is independent of the distance between electrodes, as long as the contact points on the protein can span the gap between electrodes. While there is no currently known biological role for high electronic conductance, its dependence on specific contacts has important technological implications, because no current is observed at all without at least one strongly bonded contact, so direct electrical detection is a highly selective and label-free single-molecule detection method. We demonstrate single-molecule, highly specific, label- and background free-electronic detection of IgG antibodies to HIV and Ebola viruses.


Assuntos
Condutividade Elétrica , Proteínas/química , Anticorpos Antivirais/imunologia , Técnicas Biossensoriais , Ebolavirus/imunologia , Eletrodos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Nanotecnologia
4.
Lancet ; 393(10174): 936-948, 2019 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-30777297

RESUMO

Ebolaviruses are pathogenic agents associated with a severe, potentially fatal, systemic disease in man and great apes. Four species of ebolaviruses have been identified in west or equatorial Africa. Once the more virulent forms enter the human population, transmission occurs primarily through contact with infected body fluids and can result in major epidemics in under-resourced settings. These viruses cause a disease characterised by systemic viral replication, immune suppression, abnormal inflammatory responses, major fluid and electrolyte losses, and high mortality. Despite recent progress on vaccines, and with no licensed prophylaxis or treatment available, case management is essentially supportive with management of severe multiple organ failure resulting from immune-mediated cell damage. The 2013-16 outbreak was classified by WHO as a Public Health Emergency of International Concern, which drew attention to the challenges of diseases caused by infections with ebolaviruses and questioned scientific, clinical, and societal preparation to handle future epidemics.


Assuntos
Surtos de Doenças/prevenção & controle , Ebolavirus , Doença pelo Vírus Ebola , África Ocidental/epidemiologia , Animais , Surtos de Doenças/estatística & dados numéricos , Progressão da Doença , Vacinas contra Ebola/imunologia , Ebolavirus/classificação , Ebolavirus/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/mortalidade , Doença pelo Vírus Ebola/terapia , Doença pelo Vírus Ebola/transmissão , Humanos , Cooperação Internacional
5.
Eur J Pharm Biopharm ; 136: 213-220, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30703544

RESUMO

No United States Food and Drug Administration-licensed vaccines protective against Ebola virus (EBOV) infections are currently available. EBOV vaccine candidates currently in development, as well as most currently licensed vaccines in general, require transport and storage under a continuous cold chain in order to prevent potential decreases in product efficacy. Cold chain requirements are particularly difficult to maintain in developing countries. To improve thermostability and reduce costly cold chain requirements, a subunit protein vaccine against EBOV was formulated as a glassy solid using lyophilization. Formulations of the key antigen, Ebola glycoprotein (EBOV-GP), adjuvanted with microparticulate aluminum hydroxide were prepared in liquid and lyophilized forms, and the vaccines were incubated at 40 °C for 12 weeks. Aggregation and degradation of EBOV-GP were observed in liquid formulations during the 12-week incubation period, whereas changes were minimal in lyophilized formulations. Antibody responses against EBOV-GP following three intramuscular immunizations in BALB/c mice were used to determine vaccine immunogenicity. EBOV-GP formulations were equally immunogenic in liquid and lyophilized forms. After lyophilization and reconstitution, adjuvanted vaccine formulations produced anti-EBOV-GP IgG antibody responses in mice similar to those generated against corresponding adjuvanted liquid vaccine formulations. More importantly, antibody responses in mice injected with reconstituted lyophilized vaccine formulations that had been incubated at 40 °C for 12 weeks prior to injection indicated that vaccine immunogenicity was fully retained after high-temperature storage, showing promise for future vaccine development efforts.


Assuntos
Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/química , Vacinas contra Ebola/administração & dosagem , Vacinas contra Ebola/química , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/prevenção & controle , Hidróxido de Alumínio/imunologia , Animais , Composição de Medicamentos , Estabilidade de Medicamentos , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Feminino , Liofilização , Doença pelo Vírus Ebola/imunologia , Camundongos , Camundongos Endogâmicos BALB C
6.
Rev Med Virol ; 29(3): e2036, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30706579

RESUMO

Tetherin, an interferon-inducible gene was first discovered to be an antiviral factor in 2008. A vast range of viruses, such as influenza A virus (IAV), dengue virus, Ebola virus, HIV, and RSV, have been reported to be susceptible to the antiviral activity of tetherin. Multiple reports have been published encompassing the role of tetherin in the IAV life cycle. To date, nine reports have been published regarding the role of tetherin in the IAV life cycle, with four reports supporting tetherin as an antiviral factor while five other reports suggesting no effect. To this end, this review summarizes the list of viruses currently known to be inhibited by tetherin and describes mechanisms used by viruses to overcome the antiviral potential of tetherin. Further, using IAV as disease model, we provide existing evidence in favor and against tetherin being considered as an antiviral candidate. Subsequent analysis of the experimental procedures across IAV-tetherin published reports revealed that the experimental setup (ie, cell lines, transfection reagents, and multiplicity of infection), strain-specific activity of NS1, and differing roles of NS1 in different cell lines may add up to the contributing factors leading to the discrepancies observed.


Assuntos
Antígeno 2 do Estroma da Médula Óssea/metabolismo , Interações Hospedeiro-Patógeno , Fatores Imunológicos/metabolismo , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Vírus da Dengue/imunologia , Ebolavirus/imunologia , HIV/imunologia , Humanos , Evasão da Resposta Imune , Vírus Sinciciais Respiratórios/imunologia
7.
Med Microbiol Immunol ; 208(2): 227-238, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30790057

RESUMO

Immunoinformatics has come by leaps and bounds to finding potent vaccine candidates against various pathogens. In the current study, a combination of different T (CD4+ and CD8+) and B cell epitope prediction tools was applied to find peptides containing multiple epitopes against Ebola nucleoprotein (NP) and the presentation of peptides to human leukocyte antigen (HLA) molecules was analyzed by prediction, docking and population coverage tools. Further, potential peptides were analyzed by ELISA for peptide induced IFN-γ secretion in peripheral blood mononuclear cells isolated from healthy volunteers. Six peptides were obtained after merging the overlapping multiple HLA I (CD8+) and II (CD4+) restricted T cell epitopes as well as B cell epitopes and eliminating the peptides liable to generate autoimmune and allergic response. All peptides displayed 100% conservancy in Zaire ebolavirus. In other Ebola virus species (Sudan, Bundibugyo and Taï forest) and Filoviridae members (Lloviuvirus and Margburgvirus), some peptides were found to be conserved with minor variations. Prediction tools confirmed the ability of predicted peptides to bind with diverse HLA (HLA-A, HLA-B, HLA-DP, HLA-DQ and HLA-DR) alleles. CABS-dock results displayed that the average root mean square deviation (RMSD) value was less than three in majority of cases representing strong binding affinity with HLA alleles. Population coverage analysis predicted high coverage (> 85%) for expected immune response in four continents (Africa, America, Asia and Europe). Nine out of ten blood samples exhibited enhanced IFN-γ secretion for two peptides (P2 and P3). Thus, the identified NP peptides can be considered as potential synthetic vaccine candidates against Ebola virus.


Assuntos
Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Epitopos/imunologia , Antígenos HLA/metabolismo , Doença pelo Vírus Ebola/prevenção & controle , Leucócitos Mononucleares/imunologia , Nucleoproteínas/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Biologia Computacional , Vacinas contra Ebola/genética , Ebolavirus/genética , Epitopos/genética , Antígenos HLA/genética , Voluntários Saudáveis , Humanos , Interferon gama/metabolismo , Nucleoproteínas/genética , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia
8.
BioDrugs ; 33(1): 9-14, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30604389

RESUMO

Over 40 years since the discovery of Ebola virus, the anti-Ebola virus vaccine efforts of the past 2 decades have culminated in over 12 different vaccine candidates that have been placed into a number of clinical trial phases, past and present. Of these 12 vaccines, four candidates are up to or in phase II clinical trials, and only one has completed the phase III clinical trial stage. While remarkable progress toward a national regulatory agency-approved vaccine for Ebola virus has been made, there remain unanswered questions on issues such as, but not limited to, vaccine protective immunity and duration of that immunity, and the appropriate vaccination strategy for those at risk of Ebola virus infection.


Assuntos
Vacinas contra Ebola/uso terapêutico , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Ensaios Clínicos como Assunto , Humanos
9.
Emerg Infect Dis ; 25(2): 290-298, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30666927

RESUMO

Ebola virus disease (EVD) is associated with elevated cytokine levels, and hypercytokinemia is more pronounced in fatal cases. This type of hyperinflammatory state is reminiscent of 2 rheumatologic disorders known as macrophage activation syndrome and hemophagocytic lymphohistiocytosis, which are characterized by macrophage and T-cell activation. An evaluation of 2 cohorts of patients with EVD revealed that a marker of macrophage activation (sCD163) but not T-cell activation (sCD25) was associated with severe and fatal EVD. Furthermore, substantial immunoreactivity of host tissues to a CD163-specific antibody, predominantly in areas of extensive immunostaining for Ebola virus antigens, was observed in fatal cases. These data suggest that host macrophage activation contributes to EVD pathogenesis and that directed antiinflammatory therapies could be beneficial in the treatment of EVD.


Assuntos
Antígenos CD/sangue , Antígenos de Diferenciação Mielomonocítica/sangue , Ebolavirus/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Receptores de Superfície Celular/sangue , Biomarcadores , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/virologia , Humanos , Imunoensaio , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo
10.
Nat Commun ; 10(1): 105, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631063

RESUMO

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.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Ebolavirus/imunologia , Infecções por Filoviridae/imunologia , Marburgvirus/imunologia , Doenças dos Primatas/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Ebolavirus/classificação , Ebolavirus/efeitos dos fármacos , Ebolavirus/fisiologia , Infecções por Filoviridae/terapia , Infecções por Filoviridae/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Imunoterapia/métodos , Marburgvirus/efeitos dos fármacos , Marburgvirus/fisiologia , Doenças dos Primatas/terapia , Doenças dos Primatas/virologia , Primatas , Resultado do Tratamento
11.
Cell Host Microbe ; 25(1): 39-48.e5, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30629917

RESUMO

Passive administration of monoclonal antibodies (mAbs) is a promising therapeutic approach for Ebola virus disease (EVD). However, all mAbs and mAb cocktails that have entered clinical development are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against outbreak-causing Bundibugyo virus (BDBV) and Sudan virus (SUDV). Here, we advance MBP134, a cocktail of two broadly neutralizing human mAbs, ADI-15878 from an EVD survivor and ADI-23774 from the same survivor but specificity-matured for SUDV GP binding affinity, as a candidate pan-ebolavirus therapeutic. MBP134 potently neutralized all ebolaviruses and demonstrated greater protective efficacy than ADI-15878 alone in EBOV-challenged guinea pigs. A second-generation cocktail, MBP134AF, engineered to effectively harness natural killer (NK) cells afforded additional improvement relative to its precursor in protective efficacy against EBOV and SUDV in guinea pigs. MBP134AF is an optimized mAb cocktail suitable for evaluation as a pan-ebolavirus therapeutic in nonhuman primates.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Bem-Estar do Animal , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/uso terapêutico , Antivirais , Modelos Animais de Doenças , Ebolavirus/patogenicidade , Epitopos/imunologia , Feminino , Filoviridae/imunologia , Cobaias , Doença pelo Vírus Ebola/virologia , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Recombinantes/imunologia , Resultado do Tratamento
12.
Lancet ; 393(10174): 889-898, 2019 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-30686586

RESUMO

BACKGROUND: mAb114 is a single monoclonal antibody that targets the receptor-binding domain of Ebola virus glycoprotein, which prevents mortality in rhesus macaques treated after lethal challenge with Zaire ebolavirus. Here we present expedited data from VRC 608, a phase 1 study to evaluate mAb114 safety, tolerability, pharmacokinetics, and immunogenicity. METHODS: In this phase 1, dose-escalation study (VRC 608), conducted at the US National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA), healthy adults aged 18-60 years were sequentially enrolled into three mAb114 dose groups of 5 mg/kg, 25 mg/kg, and 50 mg/kg. The drug was given to participants intravenously over 30 min, and participants were followed for 24 weeks. Participants were only enrolled into increased dosing groups after interim safety assessments. Our primary endpoints were safety and tolerability, with pharmacokinetic and anti-drug antibody assessments as secondary endpoints. We assessed safety and tolerability in all participants who received study drug by monitoring clinical laboratory data and self-report and direct clinician assessment of prespecified infusion-site symptoms 3 days after infusion and systemic symptoms 7 days after infusion. Unsolicited adverse events were recorded for 28 days. Pharmacokinetic and anti-drug antibody assessments were completed in participants with at least 56 days of data. This trial is registered with ClinicalTrials.gov, number NCT03478891, and is active but no longer recruiting. FINDINGS: Between May 16, and Sept 27, 2018, 19 eligible individuals were enrolled. One (5%) participant was not infused because intravenous access was not adequate. Of 18 (95%) remaining participants, three (17%) were assigned to the 5 mg/kg group, five (28%) to the 25 mg/kg group, and ten (55%) to the 50 mg/kg group, each of whom received a single infusion of mAb114 at their assigned dose. All infusions were well tolerated and completed over 30-37 min with no infusion reactions or rate adjustments. All participants who received the study drug completed the safety assessment of local and systemic reactogenicity. No participants reported infusion-site symptoms. Systemic symptoms were all mild and present only in four (22%) of 18 participants across all dosing groups. No unsolicited adverse events occurred related to mAb114 and one serious adverse event occurred that was unrelated to mAb114. mAb114 has linear pharmacokinetics and a half-life of 24·2 days (standard error of measurement 0·2) with no evidence of anti-drug antibody development. INTERPRETATION: mAb114 was well tolerated, showed linear pharmacokinetics, and was easily and rapidly infused, making it an attractive and deployable option for treatment in outbreak settings. FUNDING: Vaccine Research Center, US National Institute of Allergy and Infectious Diseases, and NIH.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Fatores Imunológicos/imunologia , Fatores Imunológicos/farmacocinética , Proteínas Virais/imunologia , Administração Intravenosa , Adulto , Animais , Anticorpos Monoclonais/administração & dosagem , Relação Dose-Resposta a Droga , Vacinas contra Ebola/administração & dosagem , Feminino , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Fatores Imunológicos/administração & dosagem , Macaca mulatta , Masculino , Pessoa de Meia-Idade , Adulto Jovem
13.
Diagn Pathol ; 13(1): 96, 2018 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-30567559

RESUMO

BACKGROUND: Rapid transmission and high mortality of Ebola virus disease (EVD) highlight a urgent need of large scale, convenient and effective measure for Ebola virus screening. Application of monoclonal antibodies (mAbs) are crucial for establishment of an enzyme-linked immunosorbent assay (ELISA) with high sensitivity and specificity. METHODS: The traditional cell fusion technique was used to generate a panel of hybridomas. Two mAbs were characterized by SDS-PAGE, Western blot, Indirect immunofluorescence assay (IFA). A sandwich ELISA was established using the two mAbs. The detection capability of the ELISA was evaluated. RESULTS: In the current study, we produced two murine-derived mAbs (designated as 6E3 and 3F21) towards Zaire Ebola virus glycoprotein (GP), the major viral transmembrane spike protein associated with viral attachment. It was shown that 6E3 and 3F21 recognized GP1 and GP2 subunits of the GP respectively. Furthermore, 6E3 and 3F21 bound to corresponding epitopes on GP without reciprocal topographical interpretation. Subsequently, a sandwich ELISA based on the two mAbs were established and evaluated. The detection limit was 3.6 ng/ml, with a linear range of 3.6-100 ng/ml. More importantly, Ebola virus like particles (eVLPs) were able to be detected by this established virus detection measure. CONCLUSIONS: We produced and characterized two murine-derived mAbs (designated as 6E3 and 3F21) towards Zaire Ebola virus glycoprotein (GP), and established a sandwich ELISA based on the mAbs. It was suggested that the sandwich ELISA provided an alternative method for specific and sensitive detection of Ebola virus in the field setting.


Assuntos
Anticorpos Monoclonais/imunologia , Ebolavirus/imunologia , Ensaio de Imunoadsorção Enzimática , Doença pelo Vírus Ebola/diagnóstico , Proteínas do Envelope Viral/imunologia , Animais , Especificidade de Anticorpos , Cercopithecus aethiops , Epitopos , Células HEK293 , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Hibridomas , Limite de Detecção , Camundongos Endogâmicos BALB C , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Células Vero
14.
BMC Infect Dis ; 18(1): 498, 2018 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-30285648

RESUMO

BACKGROUND: Ebolavirus and Marburgvirus are genera of the virus family Filoviridae. Filoviruses cause rare but fatal viral hemorrhagic fevers (VHFs) in remote villages of equatorial Africa with potential for regional and international spread. Point-of-care (POC) rapid diagnostic tests (RDTs) are critical for early epidemic detection, reponse and control. There are 2 RDTs for Zaire ebolavirus (EBOV), but not other Ebolavirus spp. or Marburg marburgvirus (MARV). We validate 3 conserved B cell epitopes of filovirus glycoprotein (GP) using ebola virus diseases (EVD) survivor samples, towards devising pan-filovirus RDTs. METHODS: In-silico Immuno-informatics:- (a) multiple and basic local alignments of amino-acid sequences of filovirus (4 Ebolavirus spp. & MARV) Gp1, 2 and epitope prediction and conservation analyses within context of ClusterW, BLAST-P and the immune epitope database analysis resource (IEDB-AR); alongside (b) in-vitro enzyme immuno-assays (EIAs) for SUDV Gp1, 2 antigen and host-specific antibodies (IgM and IgG) among 94 gamma irradiated EVD survivor serum and 9 negative controls. RESULTS: Linear B cell epitopes were present across the entire length of all Gp1, 2, most lying in the region between amino acids positioned 350 and 500. Three seperate epitopes 97/80_GAFFLYDRLAST, 39_YEAGEWAENCY and 500_CGLRQLANETTQALQLFLRATTELR (designated UG-Filo-Peptide- 1, 2 and 3 respectively) were conserved within all studied filovirus species Gp1, 2. Gp1, 2 host specific IgM levels were comparably low (av. ODs < 0.04 [95% CI: 0.02837 to 0.04033]) among the 9 negative controls and 57 survivor samples analyzed. Host specific IgG levels, on the other hand, were elevated (av. ODs > 1.7525 [95% CI: 0.3010 to 3.1352]) among the 92 survivor samples relative to the 9 negative controls (av. ODs < 0.2.321 [95% CI: -0.7596 to 0.5372]). Filovirus Gp1, 2 antigen was not detected [av. ODs < 0.20] within EVD survivor serum relative to recombinant protein positive controls [av. ODs = 0.50]. CONCLUSIONS: These conserved B cell epitopes of filovirus Gp1, 2 and their derivative antibodies are promising for research and development of RDTs for EVD, with potential for extension to detect MVD.


Assuntos
Ebolavirus/imunologia , Epitopos de Linfócito B/imunologia , Glicoproteínas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Sequência Conservada , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/virologia , Humanos , Imunoglobulina G , Imunoglobulina M , Doença do Vírus de Marburg/diagnóstico , Doença do Vírus de Marburg/virologia , Marburgvirus/imunologia , Kit de Reagentes para Diagnóstico , Proteínas Virais/genética , Proteínas Virais/imunologia
15.
Virol Sin ; 33(4): 323-334, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30066045

RESUMO

This study aimed to investigate the serological characteristics of Ebola virus (EBOV) infection during the late phase of the Ebola outbreak in Sierra Leone. In total, 877 blood samples from 694 suspected Ebola virus disease (EVD) cases assessed from March to December 2015, were analyzed via real-time reverse transcription polymerase chain reaction (RT-PCR) for viral RNA and enzyme-linked immunosorbent assay (ELISA) and Luminex to detect antibodies against EBOV. Viral load and EBOV-specific IgM/IgG titers displayed a declining trend during March to December 2015. Viral RNA load decreased rapidly at earlier stages after disease onset, while EBOV-specific IgM and IgG still persisted in 58.1% (18/31) and 93.5% (29/31) of the confirmed EVD patients and in 3.8% (25/663) and 17.8% (118/663) of the RNA-negative suspected patients in the later phase, respectively. Dynamic analysis of longitudinally collected samples from eight EVD patients revealed typically reversed trends of declining viral load and increasing IgM and/or IgG titers in response to the EBOV infection. The present results indicate that certain populations of Sierra Leone developed immunity to an EBOV infection in the late phase of the outbreak, providing novel insights into the risk assessment of EBOV infections among human populations.


Assuntos
Surtos de Doenças , Ebolavirus/genética , Ebolavirus/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/epidemiologia , Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática , Doença pelo Vírus Ebola/virologia , Humanos , RNA Viral/sangue , Reação em Cadeia da Polimerase em Tempo Real , Serra Leoa/epidemiologia , Fatores de Tempo , Carga Viral
16.
Proc Natl Acad Sci U S A ; 115(32): E7578-E7586, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30038008

RESUMO

The recent Ebola epidemic exemplified the importance of understanding and controlling emerging infections. Despite the importance of T cells in clearing virus during acute infection, little is known about Ebola-specific CD8+ T cell responses. We investigated immune responses of individuals infected with Ebola virus (EBOV) during the 2013-2016 West Africa epidemic in Sierra Leone, where the majority of the >28,000 EBOV disease (EVD) cases occurred. We examined T cell memory responses to seven of the eight Ebola proteins (GP, sGP, NP, VP24, VP30, VP35, and VP40) and associated HLA expression in survivors. Of the 30 subjects included in our analysis, CD8+ T cells from 26 survivors responded to at least one EBOV antigen. A minority, 10 of 26 responders (38%), made CD8+ T cell responses to the viral GP or sGP. In contrast, 25 of the 26 responders (96%) made response to viral NP, 77% to VP24 (20 of 26), 69% to VP40 (18 of 26), 42% (11 of 26) to VP35, with no response to VP30. Individuals making CD8+ T cells to EBOV VP24, VP35, and VP40 also made CD8+ T cells to NP, but rarely to GP. We identified 34 CD8+ T cell epitopes for Ebola. Our data indicate the immunodominance of the EBOV NP-specific T cell response and suggest that its inclusion in a vaccine along with the EBOV GP would best mimic survivor responses and help boost cell-mediated immunity during vaccination.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Ebolavirus/imunologia , Epidemias , Antígenos HLA/imunologia , Doença pelo Vírus Ebola/imunologia , Adolescente , Adulto , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Antígenos HLA/sangue , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Masculino , Nucleoproteínas/imunologia , Serra Leoa , Sobreviventes , Vacinação/métodos , Proteínas Virais/imunologia , Adulto Jovem
17.
Emerg Microbes Infect ; 7(1): 101, 2018 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-29872043

RESUMO

Ebolavirus vaccines based on several adenoviral vectors have been investigated in preclinical studies and clinical trials. The use of adenovirus serotype 2 as a vector for ebolavirus vaccine has not been reported. Herein, we generated rAd2-ZGP, a recombinant replication-incompetent adenovirus serotype 2 expressing codon-optimized Zaire ebolavirus glycoprotein, and evaluated its immunogenicity in mice and rhesus macaques. rAd2-ZGP induced significant antibody and cell-mediated immune responses at 2 weeks after a single immunization. The glycoprotein (GP)-specific immune responses could be further enhanced with a booster immunization. Compared to protein antigens, Zaire ebolavirus GP and Zaire ebolavirus-like particles, rAd2-ZGP could induce stronger cross-reactive antibody and cell-mediated immune responses to heterologous Sudan ebolavirus in mice and rhesus macaques. In rAd2-ZGP-immunized macaques, GP-specific CD8+ T cells could secret IFN-γ and IL-2, indicating a Th1-biased response. In adenovirus serotype 5 seropositive macaques, rAd2-ZGP could induce robust antibody and cell-mediated immune responses, suggesting that the efficacy of rAd2-ZGP is not affected by pre-existing immunity to adenovirus serotype 5. These results demonstrated that rAd2-ZGP can be considered an alternative ebolavirus vaccine for use in adenovirus serotype 5 seropositive subjects or as a sequential booster vaccine after the subjects have been immunized with a recombinant adenovirus serotype 5-based vaccine.


Assuntos
Adenoviridae/genética , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Imunidade Celular , Vacinas Virais/imunologia , Adenoviridae/metabolismo , Animais , Modelos Animais de Doenças , Ebolavirus/genética , Feminino , Glicoproteínas/administração & dosagem , Glicoproteínas/genética , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Humanos , Interferon gama/imunologia , Interleucina-2/imunologia , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , Vacinação , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
20.
PLoS Negl Trop Dis ; 12(5): e0006530, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29795572

RESUMO

BACKGROUND: Ebola virus (EBOV) caused more than 11,000 deaths during the 2013-2016 epidemic in West Africa without approved vaccines or immunotherapeutics. Despite its high lethality in some individuals, EBOV infection can produce little to no symptoms in others. A better understanding of the immune responses in individuals who experienced minimally symptomatic and asymptomatic infection could aid the development of more effective vaccines and antivirals against EBOV and related filoviruses. METHODOLOGY/PRINCIPLE FINDINGS: Between August and November 2017, blood samples were collected from 19 study participants in Lagos, Nigeria, including 3 Ebola virus disease (EVD) survivors, 10 individuals with documented close contact with symptomatic EVD patients, and 6 control healthcare workers for a cross-sectional serosurvey and T cell analysis. The Lagos samples, as well as archived serum collected from healthy individuals living in surrounding areas of the 1976 Democratic Republic of Congo (DRC) epidemic, were tested for EBOV IgG using commercial enzyme-linked immunosorbent assays (ELISAs) and Western blots. We detected antibodies in 3 out of 3 Lagos survivors and identified 2 seropositive individuals not known to have ever been infected. Of the DRC samples tested, we detected antibodies in 9 out of 71 (12.7%). To characterize the T cell responses in the Lagos samples, we developed an anthrax toxin-based enzyme-linked immunospot (ELISPOT) assay. The seropositive asymptomatic individuals had T cell responses against EBOV nucleoprotein, matrix protein, and glycoprotein 1 that were stronger in magnitude compared to the survivors. CONCLUSION/SIGNIFICANCE: Our data provide further evidence of EBOV exposure in individuals without EVD-like illness and, for the first time, demonstrate that these individuals have T cell responses that are stronger in magnitude compared to severe cases. These findings suggest that T cell immunity may protect against severe EVD, which has important implications for vaccine development.


Assuntos
Anticorpos Antivirais/sangue , Doenças Assintomáticas/epidemiologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Anticorpos Antivirais/imunologia , Antígenos de Bactérias/análise , Toxinas Bacterianas/análise , Congo/epidemiologia , Estudos Transversais , Ebolavirus/fisiologia , ELISPOT , Feminino , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/virologia , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
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