RESUMO
Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of ß2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to â¼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Sistemas CRISPR-Cas/genética , Linfócitos T/metabolismo , Diferenciação Celular , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Zika virus (ZIKV) is a mosquito-borne flavivirus with maternal infection associated with preterm birth, congenital malformations, and fetal death, and adult infection associated with Guillain-Barré syndrome. Recent widespread endemic transmission of ZIKV and the potential for future outbreaks necessitate the development of an effective vaccine. We developed a ZIKV vaccine candidate based on virus-like-particles (VLPs) generated following transfection of mammalian HEK293T cells using a plasmid encoding the pre-membrane/membrane (prM/M) and envelope (E) structural protein genes. VLPs were collected from cell culture supernatant and purified by column chromatography with yields of approximately 1-2mg/L. To promote increased particle yields, a single amino acid change of phenylalanine to alanine was made in the E fusion loop at position 108 (F108A) of the lead VLP vaccine candidate. This mutation resulted in a modest 2-fold increase in F108A VLP production with no detectable prM processing by furin to a mature particle, in contrast to the lead candidate (parent). To evaluate immunogenicity and efficacy, AG129 mice were immunized with a dose titration of either the immature F108A or lead VLP (each alum adjuvanted). The resulting VLP-specific binding antibody (Ab) levels were comparable. However, geometric mean neutralizing Ab (nAb) titers using a recombinant ZIKV reporter were significantly lower with F108A immunization compared to lead. After virus challenge, all lead VLP-immunized groups showed a significant 3- to 4-Log10 reduction in mean ZIKV RNAemia levels compared with control mice immunized only with alum, but the RNAemia reduction of 0.5 Log10 for F108A groups was statistically similar to the control. Successful viral control by the lead VLP candidate following challenge supports further vaccine development for this candidate. Notably, nAb titer levels in the lead, but not F108A, VLP-immunized mice inversely correlated with RNAemia. Further evaluation of sera by an in vitro Ab-dependent enhancement assay demonstrated that the F108A VLP-induced immune sera had a significantly higher capacity to promote ZIKV infection in FcγR-expressing cells. These data indicate that a single amino acid change in the fusion loop resulted in increased VLP yields but that the immature F108A particles were significantly diminished in their capacity to induce nAbs and provide protection against ZIKV challenge.
Assuntos
Nascimento Prematuro , Vacinas de Partículas Semelhantes a Vírus , Vacinas Virais , Infecção por Zika virus , Zika virus , Aminoácidos , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Feminino , Células HEK293 , Humanos , Recém-Nascido , Mamíferos , Camundongos , Mutação , Zika virus/genéticaRESUMO
Arthropod-borne rickettsial pathogens cause mild and severe human disease worldwide. The tick-borne pathogen Rickettsia parkeri elicits skin lesions (eschars) and disseminated disease in humans; however, inbred mice are generally resistant to infection. We report that intradermal infection of mice lacking both interferon receptors (Ifnar1-/-;Ifngr1-/-) with as few as 10 R. parkeri elicits eschar formation and disseminated, lethal disease. Similar to human infection, eschars exhibited necrosis and inflammation, with bacteria primarily found in leukocytes. Using this model, we find that the actin-based motility factor Sca2 is required for dissemination from the skin to internal organs, and the outer membrane protein OmpB contributes to eschar formation. Immunizing Ifnar1-/-;Ifngr1-/- mice with sca2 and ompB mutant R. parkeri protects against rechallenge, revealing live-attenuated vaccine candidates. Thus, Ifnar1-/-;Ifngr1-/- mice are a tractable model to investigate rickettsiosis, virulence factors, and immunity. Our results further suggest that discrepancies between mouse and human susceptibility may be due to differences in interferon signaling.
Tick bites allow disease-causing microbes, including multiple species of Rickettsia bacteria, to pass from arthropods to humans. Being exposed to Rickettsia parkeri, for example, can cause a scab at the bite site, fever, headache and fatigue. To date, no vaccine is available against any of the severe diseases caused by Rickettsia species. Modelling human infections in animals could help to understand and combat these illnesses. R. parkeri is a good candidate for such studies, as it can give insight into more severe Rickettsia infections while being comparatively safer to handle. However, laboratory mice are resistant to this species of bacteria, limiting their use as models. To explore why this is the case, Burke et al. probed whether an immune mechanism known as interferon signalling protects laboratory rodents against R. parkeri. During infection, the immune system releases molecules called interferons that stick to 'receptors' at the surface of cells, triggering defense mechanisms that help to fight off an invader. Burke et al. injected R. parkeri into the skin of mice that had or lacked certain interferon receptors, showing that animals without two specific receptors developed scabs and saw the disease spread through their body. Further investigation showed that two R. parkeri proteins, known as OmpB or Sca2, were essential for the bacteria to cause skin lesions and damage internal organs. Burke et al. then used R. parkeri that lacked OmpB or Sca2 to test whether these modified, inoffensive microbes could act as 'vaccines'. And indeed, vulnerable laboratory mice which were first exposed to the mutant bacteria were then able to survive the 'normal' version of the microbe. Together, this work reveals that interferon signalling protects laboratory mice against R. parkeri infections. It also creates an animal model that can be used to study disease and vaccination.
Assuntos
Estudos de Associação Genética , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Infecções por Rickettsia/imunologia , Animais , Medula Óssea , Feminino , Imunidade Inata , Inflamação , Listeria monocytogenes , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Rickettsia , Infecções por Rickettsia/patologia , CarrapatosRESUMO
Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the ß-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Vírus da Dengue/imunologia , Proteínas não Estruturais Virais/imunologia , Vírus do Nilo Ocidental/imunologia , Zika virus/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Reações Cruzadas , Cristalografia por Raios X , Dengue/prevenção & controle , Dengue/terapia , Endotélio/imunologia , Glicocálix/imunologia , Humanos , Camundongos , Conformação Proteica em Folha beta , Domínios Proteicos , Proteínas não Estruturais Virais/química , Febre do Nilo Ocidental/prevenção & controle , Febre do Nilo Ocidental/terapia , Infecção por Zika virus/prevenção & controle , Infecção por Zika virus/terapiaRESUMO
BACKGROUND: Acute respiratory infection is one of the main causes of morbidity in children. Some studies have suggested that pulmonary hypertension and congenital heart disease with haemodynamic repercussion increase the severity of respiratory infections, but there are few publications in developing countries. METHODS: This was a prospective cohort study evaluating the impact of pulmonary hypertension and congenital heart disease (CHD) with haemodynamic repercussion as predictors of severity in children under 5 years of age hospitalised for acute respiratory infection. RESULTS: Altogether, 217 children hospitalised for a respiratory infection who underwent an echocardiogram were evaluated; 62 children were diagnosed with CHD with haemodynamic repercussion or pulmonary hypertension. Independent predictors of admission to intensive care included: pulmonary hypertension (RR 2.14; 95% CI 1.06-4.35, p = 0.034), respiratory syncytial virus (RR 2.52; 95% CI 1.29-4.92, p = 0.006), and bacterial pneumonia (RR 3.09; 95% CI 1.65-5.81, p = 0.000). A significant difference was found in average length of hospital stay in children with the cardiovascular conditions studied (p = 0.000). CONCLUSIONS: Pulmonary hypertension and CHD with haemodynamic repercussion as well as respiratory syncytial virus and bacterial pneumonia were predictors of severity in children with respiratory infections in this study. Early recognition of cardiovascular risks in paediatric populations is necessary to lessen the impact on respiratory infections.
Assuntos
Cardiopatias Congênitas , Hipertensão Pulmonar , Infecções por Vírus Respiratório Sincicial , Infecções Respiratórias , Criança , Pré-Escolar , Colômbia/epidemiologia , Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/epidemiologia , Hemodinâmica , Humanos , Hipertensão Pulmonar/epidemiologia , Lactente , Estudos Prospectivos , Encaminhamento e Consulta , Infecções Respiratórias/complicações , Infecções Respiratórias/epidemiologia , América do SulRESUMO
Dengue is the most prevalent arboviral disease in humans and a continually increasing global public health burden. To date, there are no approved antiviral therapies against dengue virus (DENV) and the only licensed vaccine, Dengvaxia, is exclusively indicated for individuals with prior DENV infection. Endothelial hyperpermeability and vascular leak, pathogenic hallmarks of severe dengue disease, can be directly triggered by DENV non-structural protein 1 (NS1). As such, anti-NS1 antibodies can prevent NS1-triggered endothelial dysfunction in vitro and pathogenesis in vivo. Recently, goose-derived anti-DENV immunoglobulin Y (IgY) antibodies were shown to neutralize DENV and Zika virus (ZIKV) infection without adverse effects, such as antibody-dependent enhancement (ADE). In this study, we used egg yolks from DENV-immunized geese to purify IgY antibodies specific to DENV NS1 epitopes. We determined that 2 anti-NS1 IgY antibodies, NS1-1 and NS1-8, were capable of neutralizing DENV infection in vitro. In addition, these antibodies did not cross-react with the DENV Envelope (E) protein nor enhance DENV or ZIKV infection in vitro. Intriguingly, NS1-8, but not NS1-1, partially blocked NS1-induced endothelial dysfunction in vitro while neither antibody blocked binding of soluble NS1 to cells. Finally, prophylactic treatment of mice with NS1-8 conferred significant protection against lethal DENV challenge. Although further research is needed to define the mechanism of action of these antibodies, our findings highlight the potential of anti-NS1 IgY as a promising prophylactic approach against DENV infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Dengue/prevenção & controle , Imunização Passiva , Imunoglobulinas/administração & dosagem , Imunoglobulinas/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Facilitadores , Chlorocebus aethiops , Dengue/terapia , Epitopos/imunologia , Feminino , Gansos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Testes de Neutralização , Dengue Grave/imunologia , Dengue Grave/prevenção & controle , Células VeroRESUMO
The rational design of dengue virus (DENV) vaccines requires a detailed understanding of the molecular basis for antibody-mediated immunity. The durably protective antibody response to DENV after primary infection is serotype specific. However, there is an incomplete understanding of the antigenic determinants for DENV type-specific (TS) antibodies, especially for DENV serotype 3, which has only one well-studied, strongly neutralizing human monoclonal antibody (mAb). Here, we investigated the human B cell response in children after natural DENV infection in the endemic area of Nicaragua and isolated 15 DENV3 TS mAbs recognizing the envelope (E) glycoprotein. Functional epitope mapping of these mAbs and small animal prophylaxis studies revealed a complex landscape with protective epitopes clustering in at least 6-7 antigenic sites. Potently neutralizing TS mAbs recognized sites principally in E glycoprotein domains I and II, and patterns suggest frequent recognition of quaternary structures on the surface of viral particles.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Sorogrupo , Adolescente , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Criança , Pré-Escolar , Chlorocebus aethiops , Vacinas contra Dengue , Vírus da Dengue/genética , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Camundongos , Modelos Moleculares , Nicarágua , Alinhamento de Sequência , Células Vero , Proteínas do Envelope Viral/imunologia , VírionRESUMO
Arboviral diseases caused by dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses represent a major public health problem worldwide, especially in tropical areas where millions of infections occur every year. The aim of this research was to identify candidate molecules for the treatment of these diseases among the drugs currently available in the market, through in silico screening and subsequent in vitro evaluation with cell culture models of DENV and ZIKV infections. Numerous pharmaceutical compounds from antibiotics to chemotherapeutic agents presented high in silico binding affinity for the viral proteins, including ergotamine, antrafenine, natamycin, pranlukast, nilotinib, itraconazole, conivaptan and novobiocin. These five last compounds were tested in vitro, being pranlukast the one that exhibited the best antiviral activity. Further in vitro assays for this compound showed a significant inhibitory effect on DENV and ZIKV infection of human monocytic cells and human hepatocytes (Huh-7 cells) with potential abrogation of virus entry. Finally, intrinsic fluorescence analyses suggest that pranlukast may have some level of interaction with three viral proteins of DENV: envelope, capsid, and NS1. Due to its promising results, suitable accessibility in the market and reduced restrictions compared to other pharmaceuticals; the anti-asthmatic pranlukast is proposed as a drug candidate against DENV, ZIKV, and CHIKV, supporting further in vitro and in vivo assessment of the potential of this and other lead compounds that exhibited good affinity scores in silico as therapeutic agents or scaffolds for the development of new drugs against arboviral diseases.
Assuntos
Antivirais/química , Antivirais/farmacologia , Arbovírus/efeitos dos fármacos , Simulação por Computador , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos , Infecções por Arbovirus/tratamento farmacológico , Infecções por Arbovirus/virologia , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Relação Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Internalização do Vírus/efeitos dos fármacosRESUMO
Dengue virus (DENV) causes the most prevalent arboviral infection of humans, resulting in a spectrum of outcomes, ranging from asymptomatic infection to dengue fever to severe dengue characterized by vascular leakage and shock. Previously, we determined that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability, disrupts the endothelial glycocalyx layer (EGL) in vitro and triggers shedding of structural components, including sialic acid (Sia) and heparan sulfate. Here, using a murine model of dengue disease disease, we found high levels of Sia and NS1 circulating in mice with DENV-induced morbidity and lethal DENV infection. Further, we developed a liquid chromatography/mass spectrometry-based method for quantifying free Sia in serum and determined that the levels of free N-glycolylneuraminic acid were significantly higher in DENV-infected mice than in uninfected controls. These data provide additional evidence that DENV infection disrupts EGL components in vivo and warrant further research assessing Sia as a biomarker of severe dengue disease.
Assuntos
Biomarcadores/sangue , Dengue/patologia , Ácido N-Acetilneuramínico/sangue , Soro/química , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Espectrometria de Massas , Camundongos , Análise de Sobrevida , Proteínas não Estruturais Virais/sangueRESUMO
Arthropod-borne flaviviruses cause life-threatening diseases associated with endothelial hyperpermeability and vascular leak. We recently found that vascular leak can be triggered by dengue virus (DENV) non-structural protein 1 (NS1) via the disruption of the endothelial glycocalyx-like layer (EGL). However, the molecular determinants of NS1 required to trigger EGL disruption and the cellular pathway(s) involved remain unknown. Here we report that mutation of a single glycosylated residue of NS1 (N207Q) abolishes the ability of NS1 to trigger EGL disruption and induce endothelial hyperpermeability. Intriguingly, while this mutant bound to the surface of endothelial cells comparably to wild-type NS1, it was no longer internalized, suggesting that NS1 binding and internalization are distinct steps. Using endocytic pathway inhibitors and gene-specific siRNAs, we determined that NS1 was endocytosed into endothelial cells in a dynamin- and clathrin-dependent manner, which was required to trigger endothelial dysfunction in vitro and vascular leak in vivo. Finally, we found that the N207 glycosylation site is highly conserved among flaviviruses and is also essential for West Nile and Zika virus NS1 to trigger endothelial hyperpermeability via clathrin-mediated endocytosis. These data provide critical mechanistic insight into flavivirus NS1-induced pathogenesis, presenting novel therapeutic and vaccine targets for flaviviral diseases.
Assuntos
Vírus da Dengue/patogenicidade , Proteínas não Estruturais Virais/fisiologia , Substituição de Aminoácidos , Sítios de Ligação/genética , Permeabilidade Capilar , Linhagem Celular , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Endocitose/fisiologia , Células Endoteliais/fisiologia , Células Endoteliais/virologia , Glicocálix/fisiologia , Glicosilação , Células HEK293 , Humanos , Modelos Biológicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Estrutura Quaternária de Proteína , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Flaviviruses cause systemic or neurotropic-encephalitic pathology in humans. The flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein involved in viral replication, immune evasion, and vascular leakage during dengue virus infection. However, the contribution of secreted NS1 from related flaviviruses to viral pathogenesis remains unknown. Here, we demonstrate that NS1 from dengue, Zika, West Nile, Japanese encephalitis, and yellow fever viruses selectively binds to and alters permeability of human endothelial cells from lung, dermis, umbilical vein, brain, and liver in vitro and causes tissue-specific vascular leakage in mice, reflecting the pathophysiology of each flavivirus. Mechanistically, each flavivirus NS1 leads to differential disruption of endothelial glycocalyx components, resulting in endothelial hyperpermeability. Our findings reveal the capacity of a secreted viral protein to modulate endothelial barrier function in a tissue-specific manner both in vitro and in vivo, potentially influencing virus dissemination and pathogenesis and providing targets for antiviral therapies and vaccine development.
Assuntos
Vírus da Dengue/genética , Células Endoteliais/virologia , Glicocálix/virologia , Proteínas não Estruturais Virais/genética , Animais , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular , Permeabilidade da Membrana Celular , Dengue/genética , Dengue/metabolismo , Dengue/patologia , Vírus da Dengue/metabolismo , Vírus da Dengue/patogenicidade , Derme/patologia , Derme/virologia , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Células Endoteliais/patologia , Expressão Gênica , Glicocálix/química , Humanos , Fígado/patologia , Fígado/virologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Especificidade de Órgãos , Cultura Primária de Células , Veias Umbilicais/patologia , Veias Umbilicais/virologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/metabolismo , Vírus do Nilo Ocidental/patogenicidade , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/metabolismo , Vírus da Febre Amarela/patogenicidade , Zika virus/genética , Zika virus/metabolismo , Zika virus/patogenicidadeRESUMO
Endothelial dysfunction and vascular leak, pathogenic hallmarks of severe dengue disease, are directly triggered by dengue virus (DENV) nonstructural protein 1 (NS1). Previous studies have shown that immunization with NS1, as well as passive transfer of NS1-immune serum or anti-NS1 mAb, prevent NS1-mediated lethality in vivo. In this study, we evaluated the immunogenicity and protective capacity of recombinant DENV NS1 administered with cyclic dinucleotides (CDNs), potent activators of innate immune pathways and highly immunogenic adjuvants. Using both wild-type C57BL/6 mice and IFN-α/ß receptor-deficient mice, we show that NS1-CDN immunizations elicit serotype-specific and cross-reactive Ab and T cell responses. Furthermore, NS1-CDN vaccinations conferred significant homotypic and heterotypic protection from DENV2-induced morbidity and mortality. In addition, we demonstrate that high anti-NS1 Ab titers are associated with protection, supporting the role of humoral responses against DENV NS1 as correlates of protection. These findings highlight the potential of CDN-based adjuvants for inducing Ab and T cell responses and validate NS1 as an important candidate for dengue vaccine development.
Assuntos
Adjuvantes Imunológicos , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Nucleotídeos Cíclicos/imunologia , Linfócitos T/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Zika virus (ZIKV) is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected Aedes mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0%) and specificity (95.9%) versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA) of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection).IMPORTANCE The emergence of Zika virus (ZIKV) as a teratogen is a profound challenge to global public health. Molecular diagnosis of infection is straightforward during the 3-week period when patients are viremic. However, serological diagnosis thereafter of historical exposure has been confounded by cross-reactivity. Using high-density peptide arrays that tile the proteomes of a selection of flaviviruses to identify a ZIKV-specific peptide, we established two assays that enable sensitive and specific diagnosis of exposure to ZIKV. These assays may be useful in guiding clinical management of mothers at risk for potential exposure to ZIKV and enable insights into the epidemiology of ZIKV infections.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Infecção por Zika virus/diagnóstico , Arbovírus/patogenicidade , Doenças Transmissíveis Emergentes/diagnóstico , Doenças Transmissíveis Emergentes/virologia , Flavivirus/patogenicidade , RNA Viral/genética , Zika virus , Infecção por Zika virus/virologiaRESUMO
Dengue virus (DENV) is the most prevalent, medically important mosquito-borne virus. Disease ranges from uncomplicated dengue to life-threatening disease, characterized by endothelial dysfunction and vascular leakage. Previously, we demonstrated that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability in a systemic mouse model and human pulmonary endothelial cells, where NS1 disrupts the endothelial glycocalyx-like layer. NS1 also triggers release of inflammatory cytokines from PBMCs via TLR4. Here, we examined the relative contributions of inflammatory mediators and endothelial cell-intrinsic pathways. In vivo, we demonstrated that DENV NS1 but not the closely-related West Nile virus NS1 triggers localized vascular leak in the dorsal dermis of wild-type C57BL/6 mice. In vitro, we showed that human dermal endothelial cells exposed to DENV NS1 do not produce inflammatory cytokines (TNF-α, IL-6, IL-8) and that blocking these cytokines does not affect DENV NS1-induced endothelial hyperpermeability. Further, we demonstrated that DENV NS1 induces vascular leak in TLR4- or TNF-α receptor-deficient mice at similar levels to wild-type animals. Finally, we blocked DENV NS1-induced vascular leak in vivo using inhibitors targeting molecules involved in glycocalyx disruption. Taken together, these data indicate that DENV NS1-induced endothelial cell-intrinsic vascular leak is independent of inflammatory cytokines but dependent on endothelial glycocalyx components.
Assuntos
Vírus da Dengue/metabolismo , Dengue/metabolismo , Endotélio Vascular/metabolismo , Glicocálix/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Dengue/genética , Vírus da Dengue/genética , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Glicocálix/genética , Humanos , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Camundongos , Camundongos Knockout , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
Despite several decades of extensive research, the development of a highly efficacious malaria vaccine has yet to be accomplished. While the RTS,S malaria vaccine candidate shows the potential to prevent a substantial number of clinical malaria cases, significant improvements in protective efficacy are still needed. Multiple studies have shown that RTS,S induces protective antibody and CD4+ T-cell responses, but limited or negligible CD8+ T cells. In this study, we evaluated the immunogenicity and protective capacity of full-length recombinant P. falciparum circumsporozoite protein (CSP) administered with the novel cationic liposomal adjuvant system CAF09. Using newly developed transgenic rodent malaria parasites expressing the full-length P. falciparum CSP, we demonstrate that this liposome-based protein-in-adjuvant formulation is capable of inducing robust antibody and CD8+ T-cell responses that strongly inhibit parasite infection and development of liver stages, conferring durable sterilizing immunity. These findings underscore the potential of liposome-based adjuvants for inducing robust humoral and CD8+ T-cell responses and warrant further studies toward the development of novel subunit vaccine formulations with this adjuvant system.
RESUMO
Zika virus (ZIKV), a mosquito-borne flavivirus, causes devastating congenital birth defects. We isolated a human monoclonal antibody (mAb), ZKA190, that potently cross-neutralizes multi-lineage ZIKV strains. ZKA190 is highly effective in vivo in preventing morbidity and mortality of ZIKV-infected mice. NMR and cryo-electron microscopy show its binding to an exposed epitope on DIII of the E protein. ZKA190 Fab binds all 180 E protein copies, altering the virus quaternary arrangement and surface curvature. However, ZIKV escape mutants emerged in vitro and in vivo in the presence of ZKA190, as well as of other neutralizing mAbs. To counter this problem, we developed a bispecific antibody (FIT-1) comprising ZKA190 and a second mAb specific for DII of E protein. In addition to retaining high in vitro and in vivo potencies, FIT-1 robustly prevented viral escape, warranting its development as a ZIKV immunotherapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Infecção por Zika virus/terapia , Zika virus/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/química , Microscopia Crioeletrônica , Epitopos , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Alinhamento de Sequência , Proteínas do Envelope Viral/química , Zika virus/imunologiaRESUMO
Vaccines against the preerythrocytic stages of malaria are appealing because the parasite can be eliminated before disease onset and because they offer the unique possibility of targeting the parasite with both antibodies and T cells. Although the role of CD8+ T cells in preerythrocytic malaria stages is well documented, a highly effective T cell-inducing vaccine remains to be advanced. Here we report the development of a prime-boost immunization regimen with the Plasmodium falciparum circumsporozoite protein (PfCS) fused to the oligomer-forming vaccinia virus A27 protein and a modified vaccinia virus Ankara (MVA) vector expressing PfCS. This protocol induced polyfunctional CD8+ T cells with an effector memory phenotype and high PfCS antibody levels. These immune responses correlated with inhibition of liver-stage parasitemia in 80% and sterile protection in 40% of mice challenged with a transgenic P. berghei parasite line that expressed PfCS. Our findings underscore the potential of T and B cell immunization strategies for improving protective effectiveness against malaria.
Assuntos
Anticorpos Antiprotozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Malária Falciparum/prevenção & controle , Camundongos , Vacinas de DNA/imunologiaRESUMO
Recent studies have shown that immune responses against the cell-traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) can inhibit parasite infection. While these studies provide important evidence toward the development of vaccines targeting this protein, it remains unknown whether these responses could engage the Plasmodium falciparum CelTOS in vivo Using a newly developed rodent malaria chimeric parasite expressing the P. falciparum CelTOS (PfCelTOS), we evaluated the protective effect of in vivo immune responses elicited by vaccination and assessed the neutralizing capacity of monoclonal antibodies specific against PfCelTOS. Mice immunized with recombinant P. falciparum CelTOS in combination with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) or glucopyranosyl lipid adjuvant-liposome-QS21 (GLA-LSQ) adjuvant system significantly inhibited sporozoite hepatocyte infection. Notably, monoclonal antibodies against PfCelTOS strongly inhibited oocyst development of P. falciparum and Plasmodium berghei expressing PfCelTOS in Anopheles gambiae mosquitoes. Taken together, our results demonstrate that anti-CelTOS responses elicited by vaccination or passive immunization can inhibit sporozoite and ookinete infection and impair vector transmission.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Modelos Animais de Doenças , Hepatócitos/efeitos dos fármacos , Hepatócitos/parasitologia , Imunização , Imunização Passiva , Estágios do Ciclo de Vida , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Camundongos , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Proteínas RecombinantesRESUMO
Zika virus (ZIKV), a mosquito-borne flavivirus with homology to Dengue virus (DENV), has become a public health emergency. By characterizing memory lymphocytes from ZIKV-infected patients, we dissected ZIKV-specific and DENV-cross-reactive immune responses. Antibodies to nonstructural protein 1 (NS1) were largely ZIKV-specific and were used to develop a serological diagnostic tool. In contrast, antibodies against E protein domain I/II (EDI/II) were cross-reactive and, although poorly neutralizing, potently enhanced ZIKV and DENV infection in vitro and lethally enhanced DENV disease in mice. Memory T cells against NS1 or E proteins were poorly cross-reactive, even in donors preexposed to DENV. The most potent neutralizing antibodies were ZIKV-specific and targeted EDIII or quaternary epitopes on infectious virus. An EDIII-specific antibody protected mice from lethal ZIKV infection, illustrating the potential for antibody-based therapy.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/uso terapêutico , Especificidade de Anticorpos , Reações Cruzadas , Vírus da Dengue/imunologia , Modelos Animais de Doenças , Humanos , Epitopos Imunodominantes/imunologia , Memória Imunológica , Estrutura Terciária de Proteína , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Infecção por Zika virus/prevenção & controle , Infecção por Zika virus/terapiaRESUMO
Rodent transgenic parasites are useful tools for the preclinical evaluation of malaria vaccines. Over the last decade, several studies have reported the development of transgenic rodent parasites expressing P. falciparum antigens for the assessment of vaccine-induced immune responses, which traditionally have been limited to in vitro assays. However, the genetic manipulation of rodent Plasmodium species can have detrimental effects on the parasite's infectivity and development. In this chapter, we present a few guidelines for designing transfection plasmids, which should improve transfection efficiency and facilitate the generation of functional transgenic parasite strains. In addition, we provide a transfection protocol for the development of transgenic P. berghei parasites as well as practical methods to assess the viability and infectivity of these newly generated strains throughout different stages of their life cycle. These techniques should allow researchers to develop novel rodent malaria parasites expressing antigens from human malaria species and to determine whether these transgenic strains are fully infectious and thus represent stringent platforms for the in vivo evaluation of malaria vaccine candidates.