RESUMO
Previous studies demonstrated that a single intramuscular (i.m.) dose of an attenuated recombinant vesicular stomatitis virus (rVSV) vector (VesiculoVax vector platform; rVSV-N4CT1) expressing the glycoprotein (GP) from the Mayinga strain of Zaire ebolavirus (EBOV) protected nonhuman primates (NHPs) from lethal challenge with EBOV strains Kikwit and Makona. Here, we studied the immunogenicities of an expanded range of attenuated rVSV vectors expressing filovirus GP in mice. Based on data from those studies, an optimal attenuated trivalent rVSV vector formulation was identified that included rVSV vectors expressing EBOV, Sudan ebolavirus (SUDV), and the Angola strain of Marburg marburgvirus (MARV) GPs. NHPs were vaccinated with a single dose of the trivalent formulation, followed by lethal challenge 28 days later with each of the three corresponding filoviruses. At day 14 postvaccination, a serum IgG response specific for all three GPs was detected in all the vaccinated macaques. A modest and balanced cell-mediated immune response specific for each GP was also detected in a majority of the vaccinated macaques. No matter the level of total GP-specific immune response detected postvaccination, all the vaccinated macaques were protected from disease and death following lethal challenge with each of the three filoviruses. These findings indicate that vaccination with a single dose of attenuated rVSV-N4CT1 vectors each expressing a single filovirus GP may provide protection against the filoviruses most commonly responsible for outbreaks of hemorrhagic fever in sub-Saharan Africa.IMPORTANCE The West African Ebola virus Zaire outbreak in 2013 showed that the disease was not only a regional concern, but a worldwide problem, and highlighted the need for a safe and efficacious vaccine to be administered to the populace. However, other endemic pathogens, like Ebola virus Sudan and Marburg, also pose an important health risk to the public and therefore require development of a vaccine prior to the occurrence of an outbreak. The significance of our research was the development of a blended trivalent filovirus vaccine that elicited a balanced immune response when administered as a single dose and provided complete protection against a lethal challenge with all three filovirus pathogens.
Assuntos
Ebolavirus/metabolismo , Glicoproteínas/metabolismo , Doença pelo Vírus Ebola/prevenção & controle , Doença do Vírus de Marburg/prevenção & controle , Marburgvirus/metabolismo , Vesiculovirus/genética , Vacinas Virais/administração & dosagem , Animais , Anticorpos Antivirais/metabolismo , Ebolavirus/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/imunologia , Imunoglobulina G/metabolismo , Injeções Intramusculares , Macaca fascicularis , Doença do Vírus de Marburg/imunologia , Marburgvirus/imunologia , Camundongos , Vacinação , Vacinas Atenuadas , Vacinas Sintéticas , Vesiculovirus/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Vacinas Virais/imunologiaRESUMO
Acetylcholinesterase is vital for normal operation of many processes in the body. Following exposure to organophosphorus (OP) nerve agents, death can ensue without immediate medical intervention. Current therapies mitigate the cholinergic crisis caused by nerve agents but do not fully prevent long-term health concerns, for example, brain damage following seizures. Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger being investigated as an antidote for OP nerve agent poisoning. HuBChE sequesters OP nerve agent in the bloodstream preventing the nerve agent from reaching critical target organ systems. HuBChE was effective when used as both a pre-treatment and as a post-exposure therapy. HuBChE has potential for use in both military settings and to protect civilian first responders in situations where nerve agent usage is suspected. We reviewed various animal models studies evaluating the efficacy of HuBChE against nerve agent exposure, pursuant to its submission for approval under the FDA Animal Rule.
Assuntos
Antídotos/uso terapêutico , Butirilcolinesterase/uso terapêutico , Agentes Neurotóxicos/toxicidade , Animais , HumanosRESUMO
Potassium cyanide (KCN) is an inhibitor of cytochrome C oxidase causing rapid death due to hypoxia. A well-characterized model of oral KCN intoxication is needed to test new therapeutics under the Food and Drug Administration Animal Rule. Clinical signs, plasma pH and lactate concentrations, biomarkers, histopathology, and cyanide and thiocyanate toxicokinetics were used to characterize the pathology of KCN intoxication in adult and juvenile mice. The acute oral LD50s were determined to be 11.8, 11.0, 10.9, and 9.9 mg/kg in water for adult male, adult female, juvenile male, and juvenile female mice, respectively. The time to death was rapid and dose dependent; juvenile mice had a shorter mean time to death. Juvenile mice displayed a more rapid onset and higher incidence of seizures. The time to observance of respiratory signs and prostration was rapid, but mice surviving beyond 2 hours generally recovered fully within 8 hours. At doses up to the LD50, there were no gross necropsy or microscopic findings clearly attributed to administration of KCN in juvenile or adult CD-1 mice from 24 hours to 28 days post-KCN challenge. Toxicokinetic analysis indicated rapid uptake, metabolism, and clearance of plasma cyanide. Potassium cyanide caused a rapid, dose-related decrease in blood pH and increase in serum lactate concentration. An increase in fatty acid-binding protein 3 was observed at 11.5 mg/kg KCN in adult but not in juvenile mice. These studies provide a characterization of KCN intoxication in adult and juvenile mice that can be used to screen or conduct preclinical efficacy studies of potential countermeasures.
Assuntos
Modelos Animais de Doenças , Cianeto de Potássio/toxicidade , Animais , Biomarcadores/sangue , Biomarcadores/urina , Peso Corporal , Avaliação Pré-Clínica de Medicamentos , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Ácido Láctico/sangue , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos , Tiocianatos/sangue , Tiocianatos/urina , ToxicocinéticaRESUMO
The Food and Drug Administration Animal Rule requires evaluation of cardiovascular and central nervous system (CNS) effects of new therapeutics. To characterize an adult and juvenile mouse model, neurobehavioral and cardiovascular effects and pathology of a single sublethal but toxic, 8 mg/kg, oral dose of potassium cyanide (KCN) for up to 41 days postdosing were investigated. This study describes the short- and long-term sensory, motor, cognitive, and behavioral changes associated with oral dosing of a sublethal but toxic dose of KCN utilizing functional observation battery and Tier II CNS testing in adult and juvenile mice of both sexes. Selected tissues (histopathology) were evaluated for changes associated with KCN exposure with special attention to brain regions. Telemetry (adult mice only) was used to evaluate cardiovascular and temperature changes. Neurobehavioral capacity, sensorimotor responsivity or spontaneous locomotor activity, and rectal temperature were significantly reduced in adult and juvenile mice at 30 minutes post-8 mg/kg KCN dose. Immediate effects of cyanide included bradycardia, adverse electrocardiogram arrhythmic events, hypotension, and hypothermia with recovery by approximately 1 hour for blood pressure and heart rate effects and by 2 hours for body temperature. Lesions consistent with hypoxia, such as mild acute tubular necrosis in the kidneys corticomedullary junction, were the only histopathological findings and occurred at a very low incidence. The mouse KCN intoxication model indicates rapid and completely reversible effects in adult and juvenile mice following a single oral 8 mg/kg dose. Neurobehavioral and cardiovascular measurements can be used in this animal model as a trigger for treatment.
Assuntos
Comportamento Animal/efeitos dos fármacos , Sistema Cardiovascular/efeitos dos fármacos , Sistema Nervoso/efeitos dos fármacos , Cianeto de Potássio/administração & dosagem , Cianeto de Potássio/toxicidade , Administração Oral , Animais , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Eletrocardiografia , Feminino , Frequência Cardíaca/efeitos dos fármacos , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos , Neurônios/efeitos dos fármacosRESUMO
Infection with pathogenic influenza viruses is associated with intense inflammatory disease. Here, we investigated the innate immune response in mice infected with H5N1 A/Vietnam/1203/04 and with reassortant human H1N1 A/Texas/36/91 viruses containing the virulence genes hemagglutinin (HA), neuraminidase (NA) and NS1 of the 1918 pandemic virus. Inclusion of the 1918 HA and NA glycoproteins rendered a seasonal H1N1 virus capable of inducing an exacerbated host innate immune response similar to that observed for highly pathogenic A/Vietnam/1203/04 virus. Infection with 1918 HA/NA:Tx/91 and A/Vietnam/1203/04 were associated with severe lung pathology, increased cytokine and chemokine production, and significant immune cell changes, including the presence of CD11b(+)Gr-1(+) cells in the blood, lung and bone marrow. Significant differential gene expression in the lung included pathways for cell death, apoptosis, production and response to reactive oxygen radicals, as well as arginine and proline metabolism and chemokines associated with monocyte and neutrophil/granulocyte accumulation and/or activation. Arginase was produced in the lung of animals infected with A/Vietnam/1204. These results demonstrate that the innate immune cell response results in the accumulation of CD11b(+)Gr-1(+) cells and products that have previously been shown to contribute to T cell suppression.
Assuntos
Medula Óssea/imunologia , Antígeno CD11b/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Pulmão/imunologia , Infecções por Orthomyxoviridae/imunologia , Linfócitos T/imunologia , Animais , Quimiocinas/imunologia , Feminino , Perfilação da Expressão Gênica , Granulócitos/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/patogenicidade , Leucócitos/imunologia , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Virulência/imunologiaRESUMO
Pulmonary anthrax caused by exposure to inhaled Bacillus anthracis, the most lethal form of anthrax disease, is a continued military and public health concern for the United States. The vaccine AV7909, consisting of the licensed anthrax drug substance AVA adjuvanted with CpG7909, induces high levels of toxin neutralizing antibodies in healthy adults using fewer doses than AVA. This study compares the ability of one- or two-dose regimens of AV7909 to induce a protective immune response in guinea pigs challenged with a lethal dose of aerosolized B. anthracis spores 6 weeks after the last vaccine dose. The results indicated that AV7909 was less effective when delivered as a single dose compared to the two-dose regimen that resulted in dose-dependent protection against death. The toxin neutralizing assay (TNA) titer and anti-PA IgG responses were proportional to the protective efficacy, with a 50% TNA neutralizing factor (NF50) greater than 0.1 associated with survival in animals receiving two doses of vaccine. The strong protection at relatively low TNA NF50 titers in this guinea pig model supports the exploration of lower doses in clinical trials to determine if these protective levels of neutralizing antibodies can be achieved in humans; however, protection with a single dose may not be feasible.
Assuntos
Vacinas contra Antraz , Antraz , Bacillus anthracis , Adulto , Humanos , Animais , Cobaias , Antraz/prevenção & controle , Anticorpos Antibacterianos , Anticorpos Neutralizantes , Antígenos de BactériasRESUMO
The mechanisms responsible for the virulence of the highly pathogenic avian influenza (HPAI) and of the 1918 pandemic influenza virus in humans remain poorly understood. To identify crucial components of the early host response during these infections by using both conventional and functional genomics tools, we studied 34 cynomolgus macaques (Macaca fascicularis) to compare a 2004 human H5N1 Vietnam isolate with 2 reassortant viruses possessing the 1918 hemagglutinin (HA) and neuraminidase (NA) surface proteins, known conveyors of virulence. One of the reassortants also contained the 1918 nonstructural (NS1) protein, an inhibitor of the host interferon response. Among these viruses, HPAI H5N1 was the most virulent. Within 24 h, the H5N1 virus produced severe bronchiolar and alveolar lesions. Notably, the H5N1 virus targeted type II pneumocytes throughout the 7-day infection, and induced the most dramatic and sustained expression of type I interferons and inflammatory and innate immune genes, as measured by genomic and protein assays. The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection. While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.
Assuntos
Imunidade Inata/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Animais , Movimento Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Perfilação da Expressão Gênica , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Pneumopatias/patologia , Pneumopatias/virologia , Linfonodos/imunologia , Macaca , Masculino , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Taxa de Sobrevida , Linfócitos T/citologia , Linfócitos T/imunologia , Fatores de Tempo , Tropismo , Replicação ViralRESUMO
The cynomolgus monkey (Macaca fascicularis) non-human primate (NHP) is widely used for filovirus vaccine testing. To use limited BSL-4 resources efficiently and minimize NHP usage, Simon's two-stage design was adapted to screen candidate Ebola virus (EBOV) vaccines in up to six NHPs with two (optimal), three, or four NHPs in Stage 1. Using the optimal design, two NHPs were tested in Stage 1. If neither survived, the candidate was rejected. Otherwise, it was eligible for Stage 2 testing in four NHPs. Candidates advanced if four or more NHPs were protected over both stages. An 80% efficacious candidate vaccine had 88.5% probability of advancing, and a 40% efficacious candidate vaccine had 83% probability of rejection. Simon's two-stage design was used to screen 27 EBOV vaccine candidates in 43 candidate regimens that varied in dose, adjuvant, formulation, or schedule. Of the 30 candidate regimens tested using two NHPs in Stage 1, 15 were rejected, nine were withdrawn, and six were tested in Stage 2. All six tested in Stage 2 qualified to advance in the product development pipeline. Multiple regimens for the EBOV vaccines approved by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) in 2019 were tested in this program. This approach may also prove useful for screening Sudan virus (SUDV) and Marburg virus (MARV) vaccine candidates.
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The Biomedical Advanced Research and Development Authority, part of the Administration for Strategic Preparedness and Response within the U.S. Department of Health and Human Services, recognizes that the evaluation of medical countermeasures under the Animal Rule requires well-characterized and reproducible animal models that are likely to be predictive of clinical benefit. Marburg virus (MARV), one of two members of the genus Marburgvirus, is characterized by a hemorrhagic fever and a high case fatality rate for which there are no licensed vaccines or therapeutics available. This natural history study consisted of twelve cynomolgus macaques challenged with 1000 PFU of MARV Angola and observed for body weight, temperature, viremia, hematology, clinical chemistry, and coagulation at multiple time points. All animals succumbed to disease within 8 days and exhibited signs consistent with those observed in human cases, including viremia, fever, systemic inflammation, coagulopathy, and lymphocytolysis, among others. Additionally, this study determined the time from exposure to onset of disease manifestations and the time course, frequency, and magnitude of the manifestations. This study will be instrumental in the design and development of medical countermeasures to Marburg virus disease.
Assuntos
Doença do Vírus de Marburg , Marburgvirus , Contramedidas Médicas , Humanos , Animais , Marburgvirus/fisiologia , Viremia , Macaca fascicularisRESUMO
Sudan ebolavirus (SUDV) is one of four members of the Ebolavirus genus known to cause Ebola Virus Disease (EVD) in humans, which is characterized by hemorrhagic fever and a high case fatality rate. While licensed therapeutics and vaccines are available in limited number to treat infections of Zaire ebolavirus, there are currently no effective licensed vaccines or therapeutics for SUDV. A well-characterized animal model of this disease is needed for the further development and testing of vaccines and therapeutics. In this study, twelve cynomolgus macaques (Macaca fascicularis) were challenged intramuscularly with 1000 PFUs of SUDV and were followed under continuous telemetric surveillance. Clinical observations, body weights, temperature, viremia, hematology, clinical chemistry, and coagulation were analyzed at timepoints throughout the study. Death from SUDV disease occurred between five and ten days after challenge at the point that each animal met the criteria for euthanasia. All animals were observed to exhibit clinical signs and lesions similar to those observed in human cases which included: viremia, fever, dehydration, reduced physical activity, macular skin rash, systemic inflammation, coagulopathy, lymphoid depletion, renal tubular necrosis, hepatocellular degeneration and necrosis. The results from this study will facilitate the future preclinical development and evaluation of vaccines and therapeutics for SUDV.
RESUMO
The host proteome response and molecular mechanisms that drive disease in vivo during infection by a human isolate of the highly pathogenic avian influenza virus (HPAI) and 1918 pandemic influenza virus remain poorly understood. This study presents a comprehensive characterization of the proteome response in cynomolgus macaque (Macaca fascicularis) lung tissue over 7 days of infection with HPAI (the most virulent), a reassortant virus containing 1918 hemagglutinin and neuraminidase surface proteins (intermediate virulence), or a human seasonal strain (least virulent). A high-sensitivity two-dimensional liquid chromatography-tandem mass spectroscopy strategy and functional network analysis were implemented to gain insight into response pathways activated in macaques during influenza virus infection. A macaque protein database was assembled and used in the identification of 35,239 unique peptide sequences corresponding to approximately 4,259 proteins. Quantitative analysis identified an increase in expression of 400 proteins during viral infection. The abundance levels of a subset of these 400 proteins produced strong correlations with disease progression observed in the macaques, distinguishing a "core" response to viral infection from a "high" response specific to severe disease. Proteome expression profiles revealed distinct temporal response kinetics between viral strains, with HPAI inducing the most rapid response. While proteins involved in the immune response, metabolism, and transport were increased rapidly in the lung by HPAI, the other viruses produced a delayed response, characterized by an increase in proteins involved in oxidative phosphorylation, RNA processing, and translation. Proteomic results were integrated with previous genomic and pathological analysis to characterize the dynamic nature of the influenza virus infection process.
Assuntos
Modelos Animais de Doenças , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Macaca fascicularis , Proteoma/metabolismo , Vírus Reordenados/fisiologia , Animais , Feminino , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/genética , Influenza Humana/metabolismo , Pulmão/metabolismo , Pulmão/virologia , Macaca fascicularis/genética , Macaca fascicularis/metabolismo , Masculino , Proteínas/genética , Proteínas/metabolismo , Proteoma/genética , Vírus Reordenados/patogenicidade , VirulênciaRESUMO
The continuing outbreaks of ebola virus disease highlight the ongoing threat posed by filoviruses. Fortunately, licensed vaccines and therapeutics are now available for Zaire ebolavirus. However, effective medical countermeasures, such as vaccines for other filoviruses such as Sudan ebolavirus and the Marburg virus, are presently in early stages of development and, in the absence of a large outbreak, would require regulatory approval via the U.S. Food and Drug Administration (FDA) Animal Rule. The selection of an appropriate animal model and virus challenge isolates for nonclinical studies are critical aspects of the development program. Here, we have focused on the recommendation of challenge isolates for Sudan ebolavirus and Marburg virus. Based on analyses led by the Filovirus Animal and Nonclinical Group (FANG) and considerations for strain selection under the FDA Guidance for the Animal Rule, we propose prototype virus isolates for use in nonclinical challenge studies.
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An anti-Zaire Ebola virus (EBOV) glycoprotein (GP) immunoglobulin G (IgG) enzyme linked immunosorbent assay (ELISA) was developed to quantify the serum levels of anti-EBOV IgG in human and non-human primate (NHP) serum following vaccination and/or exposure to EBOV. This method was validated for testing human serum samples as previously reported. However, for direct immunobridging comparability between humans and NHPs, additional testing was warranted. First, method feasibility experiments were performed to assess cross-species reactivity and parallelism between human and NHP serum samples. During these preliminary assessments, the goat anti-human IgG secondary antibody conjugate used in the previous human validation was found to be favorably cross-reactive with NHP samples when tested at the same concentrations previously used in the validated assay for human sample testing. Further, NHP serum samples diluted in parallel with human serum when tested side-by-side in the ELISA. A subsequent NHP matrix qualification and partial validation in the anti-GP IgG ELISA were performed based on ICH and FDA guidance, to characterize assay performance for NHP test samples and supplement the previous validation for human sample testing. Based on our assessments, the anti-EBOV GP IgG ELISA method is considered suitable for the intended use of testing with both human and NHP serum samples in the same assay for immunobridging purposes.
Assuntos
Anticorpos Antivirais/sangue , Ebolavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Primatas/virologia , Animais , Estudos Transversais , Ensaio de Imunoadsorção Enzimática/normas , Estudos de Viabilidade , Humanos , Imunoglobulina G/sangue , Limite de Detecção , Padrões de ReferênciaRESUMO
Organophosphorus (OP) compounds, which include insecticides and chemical warfare nerve agents (CWNAs) such as sarin (GB) and VX, continue to be a global threat to both civilian and military populations. It is widely accepted that cholinesterase inhibition is the primary mechanism for acute OP toxicity. Disruption of cholinergic function through the inhibition of acetylcholinesterase (AChE) leads to the accumulation of the neurotransmitter acetylcholine. Excess acetylcholine at the synapse results in an overstimulation of cholinergic neurons which manifests in the common signs and symptoms of OP intoxication (miosis, increased secretions, seizures, convulsions, and respiratory failure). The primary therapeutic strategy employed in the United States to treat OP intoxication includes reactivation of inhibited AChE with the oxime pralidoxime (2-PAM) along with the muscarinic acetylcholine receptor antagonist atropine and the benzodiazepine, diazepam. CWNAs are also known to inhibit butyrylcholinesterase (BChE) without any apparent toxic effects. Therefore, BChE may be viewed as a "bioscavenger" that stoichiometrically binds CWNAs and removes them from circulation. The degree of inhibition of AChE and BChE and the effectiveness of 2-PAM are known to vary among species. Animal models are imperative for evaluating the efficacy of CWNA medical countermeasures, and a thorough characterization of available animal models is important for translating results to humans. Thus, the objective of this study was to compare the circulating levels of each of the cholinesterases as well as multiple kinetic properties (inhibition, reactivation, and aging rates) of both AChE and BChE derived from humans to AChE and BChE derived from commonly used large animal models.
Assuntos
Acetilcolinesterase/metabolismo , Antídotos/farmacologia , Butirilcolinesterase/metabolismo , Substâncias para a Guerra Química/toxicidade , Inibidores da Colinesterase/toxicidade , Reativadores da Colinesterase/farmacologia , Fatores Etários , Animais , Chlorocebus aethiops , Feminino , Proteínas Ligadas por GPI , Humanos , Cinética , Macaca fascicularis , Macaca mulatta , Masculino , Modelos Biológicos , Medição de Risco , Especificidade da Espécie , Suínos , Porco MiniaturaRESUMO
The need for an efficacious vaccine against highly pathogenic filoviruses was reinforced by the devastating 2014-2016 outbreak of Ebola virus (EBOV) disease (EVD) in Guinea, Sierra Leone, and Liberia that resulted in over 28,000 cases and over 11,300 deaths. In addition, the 2018-2020 outbreak in the Democratic Republic of the Congo currently has over 3,400 cases and over 2,200 deaths. A fully licensed vaccine and at least one other investigational vaccine are being deployed to combat this EVD outbreak. To support vaccine development and pre-clinical/clinical testing a Filovirus Animal Nonclinical Group (FANG) human anti-EBOV GP IgG ELISA was developed to measure anti-EBOV GP IgG antibodies. This ELISA is currently being used in multiple laboratories. Reported here is a characterization of an interlaboratory statistical analysis of the human anti-EBOV GP IgG ELISA as part of a collaborative study between five participating laboratories. Each laboratory used similar method protocols and reagents to measure anti-EBOV GP IgG levels in human serum samples from a proficiency panel consisting of ten serum samples created by the differential dilution of a serum sample positive for anti-GP IgG antibodies (BMIZAIRE105) with negative serum (BMI529). The total assay variability (inter- and intra-assay variability) %CVs observed at each laboratory ranged from 12.2 to 30.6. Intermediate precision (inter-assay variability) for the laboratory runs ranged from 8.9 to 21.7%CV and repeatability (intra-assay variability) %CVs ranged from 7.2 to 23.7. The estimated slope for the relationship between log10(Target Concentration) and the log10(Observed Concentration) across all five laboratories was 0.95 with a 90% confidence interval of (0.93, 0.97). Equivalence test results showed that the 90% confidence interval for the ratios for the sample-specific mean concentrations at the five individual labs to the overall laboratory consensus value were within the equivalence bounds of 0.80 to 1.25 for each laboratory and test sample, except for six test samples from Lab D, two samples from Lab B1, and one sample from Lab B2. The mean laboratory concentrations for Lab D were less than those from the other laboratories by 20% on average across the serum samples. The evaluation of the proficiency panel at these laboratories provides a limited assessment of assay precision (intermediate precision, repeatability, and total assay variability), dilutional linearity, and accuracy. This evaluation suggests that the within-laboratory performance of the anti-EBOV GP IgG ELISA as implemented at the five laboratories is consistent with the intended use of the assay based on the acceptance criteria used by laboratories that have validated the assay. However, the assessment of between-laboratory performance revealed lower observed concentrations at Lab D and greater variability in assay results at Lab B1 relative to other laboratories.
Assuntos
Anticorpos Antivirais/sangue , Ebolavirus/imunologia , Ensaio de Imunoadsorção Enzimática/normas , Doença pelo Vírus Ebola/imunologia , Proteínas Virais/imunologia , África Ocidental/epidemiologia , Animais , República Democrática do Congo/epidemiologia , Surtos de Doenças , Vacinas contra Ebola/imunologia , Vacinas contra Ebola/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Imunoglobulina G/sangue , Laboratórios , Variações Dependentes do ObservadorRESUMO
Epithelial cell migration during wound healing is regulated in part by enzymatic processing of laminin-332 (formerly LN-5), a heterodimer formed from alpha, beta, and gamma polypeptide chains. Under static conditions, laminin-332 is secreted into the extracellular matrix as a proform and has two chains processed to smaller forms, allowing it to anchor epithelial cells to the basement membrane of the dermis. During incisional wounding, laminin gamma2 chains in particular are processed to smaller sizes and function to promote epithelial sheet migration over the wound bed. The present study examines whether this same function occurs following chemical injury. The mouse ear vesicant model (MEVM) was used to follow the pathology in the ear and test whether processed laminin-332 enhances epithelial cell migration. Skin biopsies of sulfur mustard (SM) exposed ears for several time points were analyzed by histology, immunohistochemistry, real-time PCR, and Western blot analysis. SM exposure greatly increased mRNA levels for laminin-gamma2 in comparison to the other two chains. Protein production of laminin-gamma2 was upregulated, and there was an increase in the processed forms. Protein production was in excess of the amount required to form heterotrimeric laminin-332 and was associated with the migrating epithelial sheet, suggesting a potential role in wound healing for monomeric laminin-gamma2.
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Moléculas de Adesão Celular/biossíntese , Movimento Celular , Células Epiteliais/metabolismo , Laminina/biossíntese , Regulação para Cima , Cicatrização , Ferimentos e Lesões/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Orelha/patologia , Células Epiteliais/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Laminina/metabolismo , Camundongos , RNA Mensageiro/biossíntese , Ferimentos e Lesões/patologia , CalininaRESUMO
The need for an efficacious vaccine against highly pathogenic filoviruses was reinforced by the recent and devastating 2014-2016 outbreak of Ebola virus (EBOV) disease in Guinea, Sierra Leone, and Liberia that resulted in more than 10,000 casualties. Such a vaccine would need to be vetted through a U.S. Food and Drug Administration (FDA) traditional, accelerated, or Animal Rule or similar European Medicines Agency (EMA) regulatory pathway. Under the FDA Animal Rule, vaccine-induced immune responses correlating with survival of non-human primates (NHPs), or another well-characterized animal model, following lethal EBOV challenge will need to be bridged to human immune response distributions in clinical trials. When possible, species-neutral methods are ideal for detection and bridging of these immune responses, such as methods to quantify anti-EBOV glycoprotein (GP) immunoglobulin G (IgG) antibodies. Further, any method that will be used to support advanced clinical and non-clinical trials will most likely require formal validation to assess suitability prior to use. Reported here is the development, qualification, and validation of a Filovirus Animal Nonclinical Group anti-EBOV GP IgG Enzyme-Linked Immunosorbent Assay (FANG anti-EBOV GP IgG ELISA) for testing human serum samples.
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Anticorpos Antivirais/sangue , Ebolavirus , Doença pelo Vírus Ebola/sangue , Imunoglobulina G/sangue , Animais , Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas/imunologia , Haplorrinos , Humanos , Imunoglobulina G/imunologia , Libéria , Masculino , Serra Leoa , Proteínas Virais/imunologiaRESUMO
Licensure of a vaccine to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires use of the U.S. Food and Drug Administration (FDA) Animal Rule to assess vaccine efficacy as human studies are not feasible or ethical. An approach to selecting VEEV challenge strains for use under the Animal Rule was developed, taking into account Department of Defense (DOD) vaccine requirements, FDA Animal Rule guidelines, strain availability, and lessons learned from the generation of filovirus challenge agents within the Filovirus Animal Nonclinical Group (FANG). Initial down-selection to VEEV IAB and IC epizootic varieties was based on the DOD objective for vaccine protection in a bioterrorism event. The subsequent down-selection of VEEV IAB and IC isolates was based on isolate availability, origin, virulence, culture and animal passage history, known disease progression in animal models, relevancy to human disease, and ability to generate sufficient challenge material. Methods for the propagation of viral stocks (use of uncloned (wild-type), plaque-cloned, versus cDNA-cloned virus) to minimize variability in the potency of the resulting challenge materials were also reviewed. The presented processes for VEEV strain selection and the propagation of viral stocks may serve as a template for animal model development product testing under the Animal Rule to other viral vaccine programs. This manuscript is based on the culmination of work presented at the "Alphavirus Workshop" organized and hosted by the Joint Vaccine Acquisition Program (JVAP) on 15 December 2014 at Fort Detrick, Maryland, USA.
Assuntos
Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Vacinas Virais/uso terapêutico , Animais , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/virologia , Guias como Assunto , Humanos , Programas de Imunização/métodos , Programas de Imunização/normas , Virologia/métodosRESUMO
Protective antigen (PA)-specific antibody and cell-mediated immune (CMI) responses to annual and alternate booster schedules of anthrax vaccine adsorbed (AVA; BioThrax) were characterized in humans over 43 months. Study participants received 1 of 6 vaccination schedules: a 3-dose intramuscular (IM) priming series (0, 1, and 6 months) with a single booster at 42 months (4-IM); 3-dose IM priming with boosters at 18 and 42 months (5-IM); 3-dose IM priming with boosters at 12, 18, 30, and 42 months (7-IM); the 1970 licensed priming series of 6 doses (0, 0.5, 1, 6, 12, and 18 months) and two annual boosters (30 and 42 months) administered either subcutaneously (SQ) (8-SQ) or IM (8-IM); or saline placebo control at all eight time points. Antibody response profiles included serum anti-PA IgG levels, subclass distributions, avidity, and lethal toxin neutralization activity (TNA). CMI profiles included frequencies of gamma interferon (IFN-γ)- and interleukin 4 (IL-4)-secreting cells and memory B cells (MBCs), lymphocyte stimulation indices (SI), and induction of IFN-γ, IL-2, IL-4, IL-6, IL-1ß, and tumor necrosis factor alpha (TNF-α) mRNA. All active schedules elicited high-avidity PA-specific IgG, TNA, MBCs, and T cell responses with a mixed Th1-Th2 profile and Th2 dominance. Anti-PA IgG and TNA were highly correlated (e.g., month 7,r(2)= 0.86,P< 0.0001, log10 transformed) and declined in the absence of boosters. Boosters administered IM generated the highest antibody responses. Increasing time intervals between boosters generated antibody responses that were faster than and superior to those obtained with the final month 42 vaccination. CMI responses to the 3-dose IM priming remained elevated up to 43 months. (This study has been registered at ClinicalTrials.gov under registration no. NCT00119067.).
Assuntos
Vacinas contra Antraz/imunologia , Anticorpos Antibacterianos/sangue , Esquemas de Imunização , Imunização Secundária/métodos , Leucócitos Mononucleares/imunologia , Vacinas contra Antraz/administração & dosagem , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Ensaios Clínicos como Assunto , Estudos de Coortes , Citocinas/metabolismo , Humanos , Imunoglobulina G/sangue , Injeções Intramusculares , Injeções Subcutâneas , Testes de Neutralização , Placebos/administração & dosagemRESUMO
Anthrax Vaccine Adsorbed (AVA, BioThrax) is approved for use in humans as a priming series of 3 intramuscular (i.m.) injections (0, 1, 6 months; 3-IM) with boosters at 12 and 18 months, and annually thereafter for those at continued risk of infection. A reduction in AVA booster frequency would lessen the burden of vaccination, reduce the cumulative frequency of vaccine associated adverse events and potentially expand vaccine coverage by requiring fewer doses per schedule. Because human inhalation anthrax studies are neither feasible nor ethical, AVA efficacy estimates are determined using cross-species bridging of immune correlates of protection (COP) identified in animal models. We have previously reported that the AVA 3-IM priming series provided high levels of protection in non-human primates (NHP) against inhalation anthrax for up to 4 years after the first vaccination. Penalized logistic regressions of those NHP immunological data identified that anti-protective antigen (anti-PA) IgG concentration measured just prior to infectious challenge was the most accurate single COP. In the present analysis, cross-species logistic regression models of this COP were used to predict probability of survival during a 43 month study in humans receiving the current 3-dose priming and 4 boosters (12, 18, 30 and 42 months; 7-IM) and reduced schedules with boosters at months 18 and 42 only (5-IM), or at month 42 only (4-IM). All models predicted high survival probabilities for the reduced schedules from 7 to 43 months. The predicted survival probabilities for the reduced schedules were 86.8% (4-IM) and 95.8% (5-IM) at month 42 when antibody levels were lowest. The data indicated that 4-IM and 5-IM are both viable alternatives to the current AVA pre-exposure prophylaxis schedule.