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1.
Proc Natl Acad Sci U S A ; 117(7): 3768-3778, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015126

RESUMO

Antibody-based therapies are a promising treatment option for managing ebolavirus infections. Several Ebola virus (EBOV)-specific and, more recently, pan-ebolavirus antibody cocktails have been described. Here, we report the development and assessment of a Sudan virus (SUDV)-specific antibody cocktail. We produced a panel of SUDV glycoprotein (GP)-specific human chimeric monoclonal antibodies (mAbs) using both plant and mammalian expression systems and completed head-to-head in vitro and in vivo evaluations. Neutralizing activity, competitive binding groups, and epitope specificity of SUDV mAbs were defined before assessing protective efficacy of individual mAbs using a mouse model of SUDV infection. Of the mAbs tested, GP base-binding mAbs were more potent neutralizers and more protective than glycan cap- or mucin-like domain-binding mAbs. No significant difference was observed between plant and mammalian mAbs in any of our in vitro or in vivo evaluations. Based on in vitro and rodent testing, a combination of two SUDV-specific mAbs, one base binding (16F6) and one glycan cap binding (X10H2), was down-selected for assessment in a macaque model of SUDV infection. This cocktail, RIID F6-H2, provided protection from SUDV infection in rhesus macaques when administered at 50 mg/kg on days 4 and 6 postinfection. RIID F6-H2 is an effective postexposure SUDV therapy and provides a potential treatment option for managing human SUDV infection.


Assuntos
Anticorpos Antivirais/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/tratamento farmacológico , Animais , Anticorpos Monoclonais/administração & dosagem , Modelos Animais de Doenças , Ebolavirus/genética , Feminino , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Imunoterapia , Macaca mulatta , Masculino , Camundongos , Proteínas Virais/imunologia
2.
J Infect Dis ; 225(4): 733-740, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-34448858

RESUMO

BACKGROUND: Convalescent plasma has been used to treat many viral diseases including Ebola. The manufacture of a purified anti-Ebola virus (EBOV) intravenous immunoglobulin (IVIG) from pooled convalescent plasma is described in this paper. METHODS: An enzyme-linked immunosorbent assay (ELISA) targeting an EBOV surface glycoprotein antigen was used to determine the immunoglobulin titer of pooled plasma and purified anti-EBOV IVIG. Anti-EBOV IVIG was also tested in neutralization assays using a vesicular stomatitis virus pseudovirion expressing EBOV glycoprotein on its surface and with live EBOV. Finally, the efficacy of the anti-EBOV IVIG was assessed in a mouse model of EBOV infection. RESULTS: In the ELISA, the anti-EBOV IVIG was shown to have a 7-fold increase in immunoglobulin G (IgG) titer over pooled convalescent plasma. In both the pseudovirion and live virus assays, the anti-EBOV IVIG showed approximately 5- to 6-fold increased potency over pooled plasma. Anti-EBOV IVIG also significantly improved survivability in mice infected with the virus when administered concurrently or 2 days after infection. CONCLUSIONS: These data support this purified anti-EBOV IVIG merits additional investigation and clinical trials for treatment and postexposure prophylaxis of Ebola virus disease. The experience gained can be applied to manufacture hyperimmune globulins against other emerging viruses.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Animais , Anticorpos Antivirais/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Imunoglobulinas Intravenosas/uso terapêutico , Camundongos , Plasma
3.
Viruses ; 13(8)2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34452435

RESUMO

Hemorrhagic smallpox, caused by variola virus (VARV), was a rare but nearly 100% lethal human disease manifestation. Hemorrhagic smallpox is frequently characterized by secondary bacterial infection, coagulopathy, and myocardial and subendocardial hemorrhages. Previous experiments have demonstrated that intravenous (IV) cowpox virus (CPXV) exposure of macaques mimics human hemorrhagic smallpox. The goal of this experiment was to further understand the onset, nature, and severity of cardiac pathology and how it may contribute to disease. The findings support an acute late-stage myocarditis with lymphohistiocytic infiltrates in the CPXV model of hemorrhagic smallpox.


Assuntos
Vírus da Varíola Bovina/patogenicidade , Hemorragia/virologia , Miocardite/virologia , Varíola/fisiopatologia , Varíola/virologia , Doença Aguda , Animais , Modelos Animais de Doenças , Feminino , Macaca fascicularis/virologia , Masculino , Miocardite/veterinária , Varíola/complicações
4.
J Clin Invest ; 130(12): 6728-6738, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32910806

RESUMO

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the urgent need for assays that detect protective levels of neutralizing antibodies. We studied the relationship among anti-spike ectodomain (anti-ECD), anti-receptor-binding domain (anti-RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by 2 in vitro assays using convalescent plasma samples from 68 patients with COVID-19. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers and in vitro VN titers. The probability of a VN titer of ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment, was ≥80% when anti-RBD or anti-ECD titers were ≥1:1350. Of all donors, 37% lacked VN titers of ≥160. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease VN or IgG titers. Analysis of 2814 asymptomatic adults found 73 individuals with anti-ECD IgG titers of ≥1:50 and strong positive correlation with anti-RBD and VN titers. Fourteen of these individuals had VN titers of ≥1:160, and all of them had anti-RBD titers of ≥1:1350. We conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titers of ≥1:1350 may provide critical information about protection against COVID-19 disease.


Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/terapia , Imunoglobulina G , SARS-CoV-2 , Adolescente , Adulto , Idoso , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/sangue , Feminino , Humanos , Imunização Passiva , Imunoglobulina G/administração & dosagem , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Soroterapia para COVID-19
5.
PLoS Negl Trop Dis ; 10(5): e0004709, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27191161

RESUMO

Humans infected with yellow fever virus (YFV), a mosquito-borne flavivirus, can develop illness ranging from a mild febrile disease to hemorrhagic fever and death. The 17D vaccine strain of YFV was developed in the 1930s, has been used continuously since development and has proven very effective. Genetic differences between vaccine and wild-type viruses are few, yet viral or host mechanisms associated with protection or disease are not fully understood. Over the past 20 years, a number of cases of vaccine-associated disease have been identified following vaccination with 17D; these cases have been correlated with reduced immune status at the time of vaccination. Recently, several studies have evaluated T cell responses to vaccination in both humans and non-human primates, but none have evaluated the response to wild-type virus infection. In the studies described here, monocyte-derived macrophages (MDM) and dendritic cells (MoDC) from both humans and rhesus macaques were evaluated for their ability to support infection with either wild-type Asibi virus or the 17D vaccine strain and the host cytokine and chemokine response characterized. Human MoDC and MDM were also evaluated for their ability to stimulate CD4+ T cells. It was found that MoDC and MDM supported viral replication and that there were differential cytokine responses to infection with either wild-type or vaccine viruses. Additionally, MoDCs infected with live 17D virus were able to stimulate IFN-γ and IL-2 production in CD4+ T cells, while cells infected with Asibi virus were not. These data demonstrate that wild-type and vaccine YFV stimulate different responses in target antigen presenting cells and that wild-type YFV can inhibit MoDC activation of CD4+ T cells, a critical component in development of protective immunity. These data provide initial, but critical insight into regulatory capabilities of wild-type YFV in development of disease.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Linfócitos T CD4-Positivos/imunologia , Vacina contra Febre Amarela/imunologia , Vírus da Febre Amarela/imunologia , Animais , Apresentação de Antígeno , Quimiocinas/análise , Quimiocinas/imunologia , Citocinas/análise , Citocinas/imunologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/análise , Interferon gama/imunologia , Interleucina-2/análise , Interleucina-2/imunologia , Ativação Linfocitária , Macaca mulatta , Macrófagos/imunologia , Macrófagos/virologia , Vacinas Atenuadas/imunologia , Replicação Viral , Vírus da Febre Amarela/fisiologia
6.
PLoS One ; 11(11): e0166318, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27902714

RESUMO

In the fall of 2014, an international news agency reported that patients suffering from Ebola virus disease (EVD) in Liberia were treated successfully with lamivudine, an antiviral drug used to treat human immunodeficiency virus-1 and hepatitis B virus infections. According to the report, 13 out of 15 patients treated with lamivudine survived and were declared free from Ebola virus disease. In this study, the anti-Ebola virus (EBOV) activity of lamivudine and another antiretroviral, zidovudine, were evaluated in a diverse set of cell lines against two variants of wild-type EBOV. Variable assay parameters were assessed to include different multiplicities of infection, lengths of inoculation times, and durations of dosing. At a multiplicity of infection of 1, lamivudine and zidovudine had no effect on EBOV propagation in Vero E6, Hep G2, or HeLa cells, or in primary human monocyte-derived macrophages. At a multiplicity of infection of 0.1, zidovudine demonstrated limited anti-EBOV activity in Huh 7 cells. Under certain conditions, lamivudine had low anti-EBOV activity at the maximum concentration tested (320 µM). However, lamivudine never achieved greater than 30% viral inhibition, and the activity was not consistently reproducible. Combination of lamivudine and zidovudine showed no synergistic antiviral activity. Independently, a set of in vitro experiments testing lamivudine and zidovudine for antiviral activity against an Ebola-enhanced green fluorescent protein reporter virus was performed at the Centers for Disease Control and Prevention. No antiviral activity was observed for either compound. A study evaluating the efficacy of lamivudine in a guinea pig model of EVD found no survival benefit. This lack of benefit was observed despite plasma lamivudine concentrations in guinea pig of about 4 µg/ml obtained in a separately conducted pharmacokinetics study. These studies found no evidence to support the therapeutic use of lamivudine for the treatment of EVD.


Assuntos
Fármacos Anti-HIV/farmacologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Lamivudina/farmacologia , Zidovudina/farmacologia , Animais , Chlorocebus aethiops , Ebolavirus/isolamento & purificação , Cobaias , Células HeLa , Doença pelo Vírus Ebola/virologia , Humanos , Macrófagos , Projetos Piloto , Células Vero , Replicação Viral/efeitos dos fármacos
7.
Virology ; 447(1-2): 181-6, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24210113

RESUMO

To characterize T cell epitopes in monkeypox virus (MPXV) infected rhesus macaques, we utilized IFNγ Elispot assay to screen 400 predicted peptides from 20MPXV proteins. Two peptides from the F8L protein, an analog of E9L protein in vaccinia, were found to elicit CD8+ T cell responses. Prediction and in vitro MHC binding analyses suggest that one is restricted by Mamu-A1(⁎)001 and another by Mamu-A1(⁎)002. The Mamu-A1(⁎)002 epitope is completely identical in all reported sequences for variola, vaccinia, cowpox and MPXV. The Mamu-A1(⁎)001 epitope is conserved in MPXV and vaccinia, and has one residue substitution (V6>I) in some cowpox sequences and all variola sequences. Given CD8+ T-cell epitopes from E9L were also identified in humans and mice, our data suggested that F8L/E9L may be a dominant pox viral protein for CD8+ T cell responses, and may be considered as a target when designing vaccines that target pox-specific T cell responses.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Monkeypox virus/imunologia , Animais , ELISPOT , Antígenos de Histocompatibilidade Classe I/metabolismo , Interferon gama/metabolismo , Macaca mulatta , Ligação Proteica
8.
PLoS One ; 8(4): e60533, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23577120

RESUMO

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1-2×10(7) PFU) or CPXV (>10(2) PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.


Assuntos
Antígenos Virais/metabolismo , Varíola Bovina/imunologia , Monócitos/imunologia , Mpox/imunologia , Neutrófilos/imunologia , Poxviridae/imunologia , Poxviridae/fisiologia , Animais , Antígenos Virais/imunologia , Biomarcadores/sangue , Linhagem Celular , Varíola Bovina/diagnóstico , DNA Viral/sangue , Progressão da Doença , Diagnóstico Precoce , Feminino , Espaço Intracelular/imunologia , Macaca mulatta , Masculino , Mpox/diagnóstico , Monócitos/citologia , Neutrófilos/citologia , Prognóstico , Coloração e Rotulagem , Vacinas Virais/imunologia
9.
PLoS One ; 8(10): e77804, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24147080

RESUMO

Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques.


Assuntos
Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Macaca mulatta/imunologia , Macaca mulatta/virologia , Monkeypox virus/imunologia , Monkeypox virus/patogenicidade , Infecções por Poxviridae/imunologia , Animais , Antígeno CD56/metabolismo , Infecções por Poxviridae/metabolismo , Receptores CCR6/metabolismo , Receptores CCR7/metabolismo , Receptores CXCR3/metabolismo , Receptores de IgG/metabolismo
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