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1.
Emerg Microbes Infect ; 9(1): 2076-2090, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32897177

RESUMO

The current coronavirus disease 2019 (COVID-19) pandemic was the result of the rapid transmission of a highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there is no efficacious vaccine or therapeutic. Toward the development of a vaccine, here we expressed and evaluated as potential candidates four versions of the spike (S) protein using an insect cell expression system: receptor binding domain (RBD), S1 subunit, the wild-type S ectodomain (S-WT), and the prefusion trimer-stabilized form (S-2P). We showed that RBD appears as a monomer in solution, whereas S1, S-WT, and S-2P associate as homotrimers with substantial glycosylation. Cryo-electron microscopy analyses suggested that S-2P assumes an identical trimer conformation as the similarly engineered S protein expressed in 293 mammalian cells but with reduced glycosylation. Overall, the four proteins confer excellent antigenicity with convalescent COVID-19 patient sera in enzyme-linked immunosorbent assay (ELISA), yet show distinct reactivities in immunoblotting. RBD, S-WT and S-2P, but not S1, induce high neutralization titres (>3-log) in mice after a three-round immunization regimen. The high immunogenicity of S-2P could be maintained at the lowest dose (1 µg) with the inclusion of an aluminium adjuvant. Higher doses (20 µg) of S-2P can elicit high neutralization titres in non-human primates that exceed 40-times the mean titres measured in convalescent COVID-19 subjects. Our results suggest that the prefusion trimer-stabilized SARS-CoV-2 S-protein from insect cells may offer a potential candidate strategy for the development of a recombinant COVID-19 vaccine.


Assuntos
Antígenos Virais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Imunogenicidade da Vacina/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular , Infecções por Coronavirus/imunologia , Microscopia Crioeletrônica , Ensaio de Imunoadsorção Enzimática , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Peptidil Dipeptidase A/metabolismo , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Células Sf9 , Glicoproteína da Espícula de Coronavírus/genética , Spodoptera , Vacinação , Proteínas do Envelope Viral/imunologia
2.
Sci Rep ; 10(1): 15917, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985513

RESUMO

SARS-CoV-2 is the novel coronavirus responsible for the outbreak of COVID-19, a disease that has spread to over 100 countries and, as of the 26th July 2020, has infected over 16 million people. Despite the urgent need to find effective therapeutics, research on SARS-CoV-2 has been affected by a lack of suitable animal models. To facilitate the development of medical approaches and novel treatments, we compared the ACE2 receptor, and TMPRSS2 and Furin proteases usage of the SARS-CoV-2 Spike glycoprotein in human and in a panel of animal models, i.e. guinea pig, dog, cat, rat, rabbit, ferret, mouse, hamster and macaque. Here we showed that ACE2, but not TMPRSS2 or Furin, has a higher level of sequence variability in the Spike protein interaction surface, which greatly influences Spike protein binding mode. Using molecular docking simulations we compared the SARS-CoV and SARS-CoV-2 Spike proteins in complex with the ACE2 receptor and showed that the SARS-CoV-2 Spike glycoprotein is compatible to bind the human ACE2 with high specificity. In contrast, TMPRSS2 and Furin are sufficiently similar in the considered hosts not to drive susceptibility differences. Computational analysis of binding modes and protein contacts indicates that macaque, ferrets and hamster are the most suitable models for the study of inhibitory antibodies and small molecules targeting the SARS-CoV-2 Spike protein interaction with ACE2. Since TMPRSS2 and Furin are similar across species, our data also suggest that transgenic animal models expressing human ACE2, such as the hACE2 transgenic mouse, are also likely to be useful models for studies investigating viral entry.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/veterinária , Pandemias/veterinária , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/veterinária , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos/genética , Animais , Gatos , Biologia Computacional/métodos , Infecções por Coronavirus/patologia , Cricetinae , Modelos Animais de Doenças , Cães , Furões , Furina/genética , Furina/metabolismo , Cobaias , Humanos , Macaca fascicularis , Camundongos , Simulação de Acoplamento Molecular , Peptidil Dipeptidase A/genética , Pneumonia Viral/patologia , Coelhos , Ratos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
3.
J Oral Sci ; 62(4): 382-386, 2020 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-32741851

RESUMO

To investigate neuronal activity involved in responses to noxious stimuli in conscious monkeys, the animals were subjected to a task that required them to detect a small change in facial skin temperature or light (second temperature: T2, second light: V2) relative to an initial condition (T1 or V1), and to detect changes in V2 along with a heat task. Recordings were obtained from 57 neurons in the ventral premotor cortex (PMv) during the heat or light detection task. T1 neurons and T2 neurons showed increased activity only during T1 or T2, and T1/T2 neurons were activated by both T1 and T2 stimuli. T1/T2 neurons showed an increase in firing at higher T1 temperatures, whereas T1 neurons did not. About half of the non-light/heat-sensitive T1/T2 neurons showed increased firing at higher T2 temperatures, whereas T2 neurons showed no such increase. The heat responses of heat-sensitive PMv neurons were significantly suppressed when monkeys shifted their attention from heat to light. The present findings suggest that heat-sensitive PMv neurons may be involved in motor responses to noxious heat, whereas light/heat-PMv neurons may be involved in emotional and motivational aspects of pain and inappropriate motor responses to allow escape from noxious stimuli.


Assuntos
Córtex Motor , Animais , Temperatura Alta , Macaca fascicularis , Neurônios , Nociceptores
4.
Cell ; 182(3): 713-721.e9, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32778225

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 µg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Avaliação Pré-Clínica de Medicamentos/métodos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas de Produtos Inativados/uso terapêutico , Vacinas Virais/uso terapêutico , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/genética , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Feminino , Cobaias , Imunogenicidade da Vacina , Macaca fascicularis , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Pneumonia Viral/virologia , Coelhos , Ratos , Ratos Wistar , Vacinas de Produtos Inativados/efeitos adversos , Células Vero , Vacinas Virais/efeitos adversos
5.
PLoS Negl Trop Dis ; 14(8): e0008637, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790668

RESUMO

BACKGROUND: Development of vaccines and therapies against Crimean-Congo hemorrhagic fever virus (CCHFV) have been hindered by the lack of immunocompetent animal models. Recently, a lethal nonhuman primate model based on the CCHFV Hoti strain was reported. CCHFV Hoti caused severe disease in cynomolgus monkeys with 75% lethality when given by the intravenous (i.v.) route. METHODOLOGY/PRINCIPAL FINDINGS: In a series of experiments, eleven cynomologus monkeys were exposed i.v. to CCHFV Hoti and four macaques were exposed i.v. to CCHFV Afghanistan. Despite transient viremia and changes in clinical pathology such as leukopenia and thrombocytopenia developing in all 15 animals, all macaques survived to the study endpoint without developing severe disease. CONCLUSIONS/SIGNIFICANCE: We were unable to attribute differences in the results of our study versus the previous report to differences in the CCHFV Hoti stock, challenge dose, origin, or age of the macaques. The observed differences are most likely the result of the outbred nature of macaques and low animal numbers often used by necessity and for ethical considerations in BSL-4 studies. Nonetheless, while we were unable to achieve severe disease or lethality, the CCHFV Hoti and Afghanistan macaque models are useful for screening medical countermeasures using biomarkers including viremia and clinical pathology to assess efficacy.


Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Febre Hemorrágica da Crimeia/virologia , Viremia , Afeganistão , Animais , Quimiocinas/sangue , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Febre Hemorrágica da Crimeia/patologia , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout
6.
Signal Transduct Target Ther ; 5(1): 157, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814760

RESUMO

Identification of a suitable nonhuman primate (NHP) model of COVID-19 remains challenging. Here, we characterized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in three NHP species: Old World monkeys Macaca mulatta (M. mulatta) and Macaca fascicularis (M. fascicularis) and New World monkey Callithrix jacchus (C. jacchus). Infected M. mulatta and M. fascicularis showed abnormal chest radiographs, an increased body temperature and a decreased body weight. Viral genomes were detected in swab and blood samples from all animals. Viral load was detected in the pulmonary tissues of M. mulatta and M. fascicularis but not C. jacchus. Furthermore, among the three animal species, M. mulatta showed the strongest response to SARS-CoV-2, including increased inflammatory cytokine expression and pathological changes in the pulmonary tissues. Collectively, these data revealed the different susceptibilities of Old World and New World monkeys to SARS-CoV-2 and identified M. mulatta as the most suitable for modeling COVID-19.


Assuntos
Betacoronavirus/patogenicidade , Callithrix/virologia , Infecções por Coronavirus/epidemiologia , Modelos Animais de Doenças , Macaca fascicularis/virologia , Macaca mulatta/virologia , Pandemias , Pneumonia Viral/epidemiologia , Animais , Anticorpos Antivirais/biossíntese , Betacoronavirus/imunologia , Temperatura Corporal , Peso Corporal , Callithrix/imunologia , Infecções por Coronavirus/diagnóstico por imagem , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Citocinas/biossíntese , Citocinas/classificação , Citocinas/imunologia , Suscetibilidade a Doenças , Feminino , Humanos , Pulmão/diagnóstico por imagem , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Macaca fascicularis/imunologia , Macaca mulatta/imunologia , Masculino , Pneumonia Viral/diagnóstico por imagem , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Especificidade da Espécie , Tomografia Computadorizada por Raios X , Carga Viral , Replicação Viral
7.
Transplantation ; 104(8): 1580-1590, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32732835

RESUMO

BACKGROUND: Although short-term outcomes for liver transplantation have improved, patient and graft survival are limited by infection, cancer, and other complications of immunosuppression. Rapid induction of tolerance after liver transplantation would decrease these complications, improving survival and quality of life. Tolerance to kidneys, but not thoracic organs or islets, has been achieved in nonhuman primates and humans through the induction of transient donor chimerism. Since the liver is considered to be tolerogenic, we tested the hypothesis that the renal transplant transient chimerism protocol would induce liver tolerance. METHODS: Seven cynomolgus macaques received immune conditioning followed by simultaneous donor bone marrow and liver transplantation. The more extensive liver surgery required minor adaptations of the kidney protocol to decrease complications. All immunosuppression was discontinued on postoperative day (POD) 28. Peripheral blood chimerism, recipient immune reconstitution, liver function tests, and graft survival were determined. RESULTS: The level and duration of chimerism in liver recipients were comparable to those previously reported in renal transplant recipients. However, unlike in the kidney model, the liver was rejected soon after immunosuppression withdrawal. Rejection was associated with proliferation of recipient CD8 T effector cells in the periphery and liver, increased serum interleukin (IL)-6 and IL-2, but peripheral regulatory T cell (Treg) numbers did not increase. Antidonor antibody was also detected. CONCLUSIONS: These data show the transient chimerism protocol does not induce tolerance to livers, likely due to greater CD8 T cell responses than in the kidney model. Successful tolerance induction may depend on greater control or deletion of CD8 T cells in this model.


Assuntos
Transplante de Medula Óssea/efeitos adversos , Rejeição de Enxerto/prevenção & controle , Transplante de Fígado/efeitos adversos , Quimeras de Transplante/imunologia , Condicionamento Pré-Transplante/métodos , Aloenxertos/imunologia , Animais , Medula Óssea/imunologia , Transplante de Medula Óssea/métodos , Modelos Animais de Doenças , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Humanos , Fígado/imunologia , Transplante de Fígado/métodos , Macaca fascicularis , Linfócitos T Citotóxicos/imunologia , Tolerância ao Transplante , Transplante Homólogo/efeitos adversos
8.
Cell ; 182(5): 1271-1283.e16, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32795413

RESUMO

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.


Assuntos
RNA Mensageiro/genética , RNA Viral/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Chlorocebus aethiops , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Feminino , Células HEK293 , Células HeLa , Humanos , Imunogenicidade da Vacina , Injeções Intramusculares , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos ICR , Nanopartículas/química , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Th1/imunologia , Potência de Vacina , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
9.
Proc Natl Acad Sci U S A ; 117(33): 20190-20197, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747564

RESUMO

Arboviruses maintain high mutation rates due to lack of proofreading ability of their viral polymerases, in some cases facilitating adaptive evolution and emergence. Here we show that, just before its 2013 spread to the Americas, Zika virus (ZIKV) underwent an envelope protein V473M substitution (E-V473M) that increased neurovirulence, maternal-to-fetal transmission, and viremia to facilitate urban transmission. A preepidemic Asian ZIKV strain (FSS13025 isolated in Cambodia in 2010) engineered with the V473M substitution significantly increased neurovirulence in neonatal mice and produced higher viral loads in the placenta and fetal heads in pregnant mice. Conversely, an epidemic ZIKV strain (PRVABC59 isolated in Puerto Rico in 2015) engineered with the inverse M473V substitution reversed the pathogenic phenotypes. Although E-V473M did not affect oral infection of Aedes aegypti mosquitoes, competition experiments in cynomolgus macaques showed that this mutation increased its fitness for viremia generation, suggesting adaptive evolution for human viremia and hence transmission. Mechanistically, the V473M mutation, located at the second transmembrane helix of the E protein, enhances virion morphogenesis. Overall, our study revealed E-V473M as a critical determinant for enhanced ZIKV virulence, intrauterine transmission during pregnancy, and viremia to facilitate urban transmission.


Assuntos
Epidemias , Proteínas do Envelope Viral/genética , Infecção por Zika virus/virologia , Zika virus/genética , Zika virus/patogenicidade , Animais , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Filogenia , Gravidez , Carga Viral , Virulência , Zika virus/fisiologia , Infecção por Zika virus/epidemiologia
10.
PLoS Pathog ; 16(7): e1008413, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730321

RESUMO

Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB), regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model of human TB, we compared radiographic, immunologic and microbiologic characteristics of early (subclinical) reactivation of latent M. tuberculosis (Mtb) infection among animals subsequently infected with simian immunodeficiency virus (SIV) or who underwent anti-CD4 depletion by a depletion antibody. CD4 depleted animals had significantly fewer CD4 T cells within granulomas compared to Mtb/SIV co-infected and Mtb-only control animals. After 2 months of treatment, subclinical reactivation occurred at similar rates among CD4 depleted (5 of 7 animals) and SIV infected animals (4 of 8 animals). However, SIV-induced reactivation was associated with more dissemination of lung granulomas that were permissive to Mtb growth resulting in greater bacterial burden within granulomas compared to CD4 depleted reactivators. Granulomas from Mtb/SIV animals displayed a more robust T cell activation profile (IFN-α, IFN-γ, TNF, IL-17, IL-2, IL-10, IL-4 and granzyme B) compared to CD4 depleted animals and controls though these effectors did not protect against reactivation or dissemination, but instead may be related to increased viral and/or Mtb antigens. SIV replication within the granuloma was associated with reactivation, greater overall Mtb growth and reduced Mtb killing resulting in greater overall Mtb burden. These data support that SIV disrupts protective immune responses against latent Mtb infection beyond the loss of CD4 T cells, and that synergy between SIV and Mtb occurs within granulomas.


Assuntos
Coinfecção/imunologia , Tuberculose Latente/imunologia , Tuberculose Latente/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Ativação Viral/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Granuloma/virologia , Hospedeiro Imunocomprometido/imunologia , Macaca fascicularis , Mycobacterium tuberculosis/imunologia , Vírus da Imunodeficiência Símia/imunologia
11.
Nat Commun ; 11(1): 3369, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632153

RESUMO

Induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons are an expected source for cell-based therapies for Parkinson's disease (PD). The regulatory criteria for the clinical application of these therapies, however, have not been established. Here we show the results of our pre-clinical study, in which we evaluate the safety and efficacy of dopaminergic progenitors (DAPs) derived from a clinical-grade human iPSC line. We confirm the characteristics of DAPs by in vitro analyses. We also verify that the DAP population include no residual undifferentiated iPSCs or early neural stem cells and have no genetic aberration in cancer-related genes. Furthermore, in vivo studies using immunodeficient mice reveal no tumorigenicity or toxicity of the cells. When the DAPs are transplanted into the striatum of 6-OHDA-lesioned rats, the animals show behavioral improvement. Based on these results, we started a clinical trial to treat PD patients in 2018.


Assuntos
Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/transplante , Doença de Parkinson/terapia , Transplante de Células-Tronco/métodos , Animais , Diferenciação Celular/genética , Linhagem Celular , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Avaliação de Resultados em Cuidados de Saúde/métodos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Ratos Nus , Transplante Heterólogo
12.
Fertil Steril ; 114(1): 33-43, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32622411

RESUMO

OBJECTIVE: To identify cell types in the male and female reproductive systems at risk for SARS-CoV-2 infection because of the expression of host genes and proteins used by the virus for cell entry. DESIGN: Descriptive analysis of transcriptomic and proteomic data. SETTING: Academic research department and clinical diagnostic laboratory. PATIENT(S): Not applicable (focus was on previously generated gene and protein expression data). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Identification of cell types coexpressing the key angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) genes and proteins as well as other candidates potentially involved in SARS-CoV-2 cell entry. RESULT(S): On the basis of single-cell RNA sequencing data, coexpression of ACE2 and TMPRSS2 was not detected in testicular cells, including sperm. A subpopulation of oocytes in nonhuman primate ovarian tissue was found to express ACE2 and TMPRSS2, but coexpression was not observed in ovarian somatic cells. RNA expression of TMPRSS2 in 18 samples of human cumulus cells was shown to be low or absent. There was general agreement between publicly available bulk RNA and protein datasets in terms of ACE2 and TMPRSS2 expression patterns in testis, ovary, endometrial, and placental cells. CONCLUSION(S): These analyses suggest that SARS-CoV-2 infection is unlikely to have long-term effects on male and female reproductive function. Although the results cannot be considered definitive, they imply that procedures in which oocytes are collected and fertilized in vitro are associated with very little risk of viral transmission from gametes to embryos and may indeed have the potential to minimize exposure of susceptible reproductive cell types to infection in comparison with natural conception.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Fertilidade/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Pneumonia Viral/metabolismo , Reprodução/fisiologia , Internalização do Vírus , Adolescente , Adulto , Animais , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Feminino , Humanos , Macaca fascicularis , Masculino , Ovário/citologia , Ovário/metabolismo , Ovário/virologia , Pandemias , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Gravidez , Proteômica/métodos , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Testículo/citologia , Testículo/metabolismo , Testículo/virologia , Transcriptoma/fisiologia , Adulto Jovem
13.
PLoS Negl Trop Dis ; 14(7): e0008459, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667913

RESUMO

Rabies, caused by RNA viruses in the Genus Lyssavirus, is the most fatal of all infectious diseases. This neglected zoonosis remains a major public health problem in developing countries, causing the death of an estimated 25,000-159,000 people each year, with more than half of them in children. The high incidence of human rabies in spite of effective vaccines is mainly linked to the lack of compliance with the complicated administration schedule, inadequacies of the community public health system for local administration by the parenteral route and the overall costs of the vaccine. The goal of our work was the development of a simple, affordable and effective vaccine strategy to prevent human rabies virus infection. This next generation vaccine is based on a replication-defective chimpanzee adenovirus vector belonging to group C, ChAd155-RG, which encodes the rabies glycoprotein (G). We demonstrate here that a single dose of this vaccine induces protective efficacy in a murine model of rabies challenge and elicits strong and durable neutralizing antibody responses in vaccinated non-human primates. Importantly, we demonstrate that one dose of a commercial rabies vaccine effectively boosts the neutralizing antibody responses induced by ChAd155-RG in vaccinated monkeys, showing the compatibility of the novel vectored vaccine with the current post-exposure prophylaxis in the event of rabies virus exposure. Finally, we demonstrate that antibodies induced by ChAd155-RG can also neutralize European bat lyssaviruses 1 and 2 (EBLV-1 and EBLV-2) found in bat reservoirs.


Assuntos
Adenovirus dos Símios/genética , Vacinas Antirrábicas/imunologia , Raiva/prevenção & controle , Animais , Antígenos Virais , Feminino , Vetores Genéticos/genética , Humanos , Macaca fascicularis , Camundongos , Pan troglodytes/virologia , Profilaxia Pós-Exposição , Coelhos , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Sorogrupo , Vacinação , Vacinas Sintéticas/imunologia , Zoonoses
14.
Nature ; 585(7826): 584-587, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32698191

RESUMO

Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic and no antiviral drug or vaccine is yet available for the treatment of this disease1-3. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes COVID-19-worldwide but there is no definitive evidence that HCQ is effective for treating COVID-194-7. Here we evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in African green monkey kidney cells (Vero E6) but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to a placebo treatment, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor the combination of HCQ and AZTH showed a significant effect on viral load in any of the analysed tissues. When the drug was used as a pre-exposure prophylaxis treatment, HCQ did not confer protection against infection with SARS-CoV-2. Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral drug for the treatment of COVID-19 in humans.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Hidroxicloroquina/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Animais , Azitromicina/farmacologia , Azitromicina/uso terapêutico , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/fisiopatologia , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Humanos , Hidroxicloroquina/farmacocinética , Hidroxicloroquina/farmacologia , Técnicas In Vitro , Cinética , Macaca fascicularis , Masculino , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/fisiopatologia , Profilaxia Pré-Exposição , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/virologia , Fatores de Tempo , Falha de Tratamento , Células Vero , Carga Viral/efeitos dos fármacos
15.
PLoS One ; 15(7): e0235815, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673351

RESUMO

Monoclonal antibodies (mAbs) for therapeutic applications should be as similar to native human antibodies as possible to minimize their immunogenicity in patients. Several transgenic animal platforms are available for the generation of fully human mAbs. Attributes such as specificity, efficacy and Chemistry, Manufacturing and Controls (CMC) developability of antibodies against a specific target are typically established for antibodies obtained from one platform only. In this study, monoclonal antibodies (mAbs) cross-reactive against human and cynomolgus LAMP1 were derived from the human immunoglobulin transgenic TRIANNI mouse and OmniChicken® platforms and assessed for their specificity, sequence diversity, ability to bind to and internalize into tumor cells, expected immunogenicity and CMC developability. Our results show that the two platforms were complementary at providing a large diversity of mAbs with respect to epitope coverage and antibody sequence diversity. Furthermore, most antibodies originating from either platform exhibited good manufacturability characteristics.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana Associadas ao Lisossomo/imunologia , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/química , Galinhas , Células HEK293 , Humanos , Imunização , Macaca fascicularis , Camundongos , Modelos Moleculares
16.
Vet Res Commun ; 44(3-4): 101-110, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32651761

RESUMO

The outbreak of the SARS-CoV-2 in mainland China with subsequent human to human transmission worldwide had taken up the shape of a devastating pandemic. The ability of the virus to infect multiple species other than humans has currently been reported in experimental conditions. Non-human primates, felines, ferrets, rodents and host of other animals could previously be infected in experimental conditions with SARS-CoV and recently with SARS-CoV-2, both virus using Angiotensin-converting-enzyme 2 receptor for cellular entry. The variations in sequence homology of ACE2 receptor across species is identified as one of the factors determining virulence and pathogenicity in animals. The infection in experimental animals with SARS-CoV or SARS-CoV-2 on most occasions are asymptomatic, however, the virus could multiply within the respiratory tract and extra-pulmonary organs in most of the species. Here, we discuss about the pathogenicity, transmission, variations in angiotensin-converting-enzyme 2 receptor-binding across species and host pathogen interactions of SARS and SARS-CoV-2 in laboratory animals used in research.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/veterinária , Interações Hospedeiro-Patógeno , Pandemias/veterinária , Pneumonia Viral/veterinária , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/veterinária , Animais , Callithrix/virologia , Gatos/virologia , Galinhas/virologia , Quirópteros/virologia , Chlorocebus aethiops/virologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Cricetinae/virologia , Furões/virologia , Macaca fascicularis/virologia , Macaca mulatta/virologia , Camundongos , Camundongos Endogâmicos/virologia , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Roedores/virologia , Síndrome Respiratória Aguda Grave/transmissão , Síndrome Respiratória Aguda Grave/virologia , Suínos/virologia
17.
MAbs ; 12(1): 1770028, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32486889

RESUMO

Many therapeutic monoclonal antibodies (mAbs) were initially developed for intravenous (IV) administration. As a means to improve mAb drug-ability and the patient experience, subcutaneous (SC) administration is an increasingly important delivery route for mAbs. Unlike IV administration, bioavailability limitations for antibodies have been reported following SC injection and can dictate whether a mAb is administered via this parenteral route. The SC bioavailability of antibodies has been difficult to predict, and it can be variable and partial, with values ranging from ~50% to 100%. The mechanisms leading to the incomplete bioavailability of some mAbs relative to others are not well understood. There are some limited data that suggest the physiochemical properties inherent to a mAb can contribute to its SC absorption, bioavailability, and in vivo fate. In this study, we evaluated the integrated influence of multiple mAb physiochemical factors on the SC absorption and bioavailability of six humanized mAbs in both rats and cynomolgus monkeys. We demonstrate the physiochemical properties of mAbs are critical to their rate and extent of SC absorption. The combination of high positive charge and hydrophobic interaction significantly reduced the rate of the evaluated mAb's SC absorption and bioavailability. Reduction or balancing of both these attributes via re-engineering the mAbs restored desirable properties of the molecules assessed. This included reduced association with SC tissue, improvements in mAb absorption from the SC space and overall SC bioavailability. Our findings point to the importance of evaluating the relative balance between various physiochemical factors, including charge, hydrophobicity, and stability, to improve the SC drug-ability of mAbs for selecting or engineering mAbs with enhanced in vivo absorption and bioavailability following SC administration.


Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Físico-Química/métodos , Animais , Anticorpos Monoclonais Humanizados/química , Bioengenharia , Disponibilidade Biológica , Desenvolvimento de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Injeções Subcutâneas , Macaca fascicularis , Ligação Proteica , Estabilidade Proteica , Ratos , Absorção Subcutânea
18.
J Pharmacol Sci ; 143(4): 272-280, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32499095

RESUMO

We analyzed the effects of intravenously as well as orally administered moxifloxacin on the pharmacokinetic and electrocardiographic variables along with its torsadogenic action using the chronic atrioventricular block cynomolgus monkeys with a cross-over design. Initially, moxifloxacin was intravenously administered in doses of 60 mg/kg/2 h, 60 mg/kg/1 h and 105 mg/kg/1.75 h with an interval of >1 week (n = 3), which provided Cmax of 19.7, 25.4 and 37.8 µg/mL, and induced torsade de pointes in 1, 0 and 3 out of 3 animals, respectively. Next, moxifloxacin was orally administered in doses of 10, 30 and 100 mg/kg with an interval of >1 week (n = 6), which provided Cmax of 1.8, 4.2 and 8.9 µg/mL, and induced torsade de pointes in 0, 0 and 2 out of 6 animals, respectively. A close analysis of pharmacokinetic and electrocardiographic variables indicates that torsade de pointes was induced in animals that had experienced larger systemic exposure of moxifloxacin and/or greater peak QTcF, although Cmax by itself did not necessarily reflect the incidence of torsade de pointes when its administration route was different. These findings may provide a basic guide how to use moxifloxacin in safe for patients with labile repolarization process.


Assuntos
Bloqueio Atrioventricular/tratamento farmacológico , Moxifloxacina/administração & dosagem , Moxifloxacina/efeitos adversos , Torsades de Pointes/induzido quimicamente , Administração Oral , Animais , Estudos Cross-Over , Eletrocardiografia , Infusões Intravenosas , Macaca fascicularis , Moxifloxacina/farmacocinética , Moxifloxacina/farmacologia
19.
PLoS Pathog ; 16(6): e1008592, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555740

RESUMO

The neuraminidase (NA) inhibitor (NAI) oseltamivir (OST) is the most widely used influenza antiviral drug. Several NA amino acid substitutions are reported to reduce viral susceptibility to OST in in vitro assays. However, whether there is a correlation between the level of reduction in susceptibility in vitro and the efficacy of OST against these viruses in vivo is not well understood. In this study, a ferret model was utilised to evaluate OST efficacy against circulating influenza A and B viruses with a range of in vitro generated 50% inhibitory concentrations (IC50) values for OST. OST efficacy against an A(H1N1)pdm09 and an A(H1N1)pdm09 virus with the H275Y substitution in neuraminidase was also tested in the macaque model. The results from this study showed that OST had a significant impact on virological parameters compared to placebo treatment of ferrets infected with wild-type influenza A viruses with normal IC50 values (~1 nM). However, this efficacy was lower against wild-type influenza B and other viruses with higher IC50 values. Differing pathogenicity of the viruses made evaluation of clinical parameters difficult, although some effect of OST in reducing clinical signs was observed with influenza A(H1N1) and A(H1N1)pdm09 (H275Y) viruses. Viral titres in macaques were too low to draw conclusive results. Analysis of the ferret data revealed a correlation between IC50 and OST efficacy in reducing viral shedding but highlighted that the current WHO guidelines/criteria for defining normal, reduced or highly reduced inhibition in influenza B viruses based on in vitro data are not well aligned with the low in vivo OST efficacy observed for both wild-type influenza B viruses and those with reduced OST susceptibility.


Assuntos
Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza B/metabolismo , Infecções por Orthomyxoviridae , Oseltamivir/farmacologia , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Furões , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza B/genética , Macaca fascicularis , Macrolídeos , Masculino , Mutação de Sentido Incorreto , Neuraminidase/genética , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia
20.
PLoS One ; 15(6): e0234038, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492075

RESUMO

Extracellular adenosine triphosphate (eATP) released by damaged cells, and its purinergic receptors, comprise a crucial signaling network after injury. Purinergic receptor P2X7 (P2RX7), a major driver of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and IL-1ß processing, has been shown to play a role in liver injury in murine diet- and chemically-induced liver injury models. It is unclear, however, whether P2RX7 plays a role in non-alcoholic steatohepatitis (NASH) and which cell type is the main target of P2RX7 pharmacological inhibition. Here, we report that P2RX7 is expressed by infiltrating monocytes and resident Kupffer cells in livers from NASH-affected individuals. Using primary isolated human cells, we demonstrate that P2RX7 expression in CD14+ monocytes and Kupffer cells primarily mediates IL-1ß release. In addition, we show that pharmacological inhibition of P2RX7 in monocytes and Kupffer cells, blocks IL-1ß release, reducing hepatocyte caspase 3/7 activity, IL-1ß-mediated CCL2 and CCL5 chemokine gene expression and secretion, and hepatic stellate cell (HSC) procollagen secretion. Consequently, in a chemically-induced nonhuman primate model of liver fibrosis, treatment with a P2RX7 inhibitor improved histological characteristics of NASH, protecting from liver inflammation and fibrosis. Taken together, these findings underscore the critical role of P2RX7 in the pathogenesis of NASH and implicate P2RX7 as a promising therapeutic target for the management of this disease.


Assuntos
Inflamação/prevenção & controle , Cirrose Hepática/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Receptores Purinérgicos P2X7/metabolismo , Animais , Caspase 3/metabolismo , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Inflamação/patologia , Interleucina-1beta/metabolismo , Macrófagos do Fígado/citologia , Macrófagos do Fígado/efeitos dos fármacos , Macrófagos do Fígado/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Macaca fascicularis , Masculino , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Pró-Colágeno/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética
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