RESUMO
Emerging studies indicate that cooperation between neurons and immune cells regulates antimicrobial immunity, inflammation and tissue homeostasis. For example, a neuronal rheostat provides excitatory or inhibitory signals that control the functions of tissue-resident group 2 innate lymphoid cells (ILC2s) at mucosal barrier surfaces1-4. ILC2s express NMUR1, a receptor for neuromedin U (NMU), which is a prominent cholinergic neuropeptide that promotes ILC2 responses5-7. However, many functions of ILC2s are shared with adaptive lymphocytes, including the production of type 2 cytokines8,9 and the release of tissue-protective amphiregulin (AREG)10-12. Consequently, there is controversy regarding whether innate lymphoid cells and adaptive lymphocytes perform redundant or non-redundant functions13-15. Here we generate a new genetic tool to target ILC2s for depletion or gene deletion in the presence of an intact adaptive immune system. Transgenic expression of iCre recombinase under the control of the mouse Nmur1 promoter enabled ILC2-specific deletion of AREG. This revealed that ILC2-derived AREG promotes non-redundant functions in the context of antiparasite immunity and tissue protection following intestinal damage and inflammation. Notably, NMU expression levels increased in inflamed intestinal tissues from both mice and humans, and NMU induced AREG production in mouse and human ILC2s. These results indicate that neuropeptide-mediated regulation of non-redundant functions of ILC2s is an evolutionarily conserved mechanism that integrates immunity and tissue protection.
Assuntos
Imunidade Inata , Mucosa Intestinal , Linfócitos , Neuropeptídeos , Animais , Humanos , Camundongos , Citocinas/imunologia , Citocinas/metabolismo , Imunidade Inata/imunologia , Inflamação/imunologia , Inflamação/parasitologia , Inflamação/patologia , Linfócitos/imunologia , Neuropeptídeos/metabolismo , Neuropeptídeos/fisiologia , Anfirregulina , Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Mucosa Intestinal/patologiaRESUMO
MEF2B encodes a transcriptional activator and is mutated in â¼11% of diffuse large B cell lymphomas (DLBCLs) and â¼12% of follicular lymphomas (FLs). Here we found that MEF2B directly activated the transcription of the proto-oncogene BCL6 in normal germinal-center (GC) B cells and was required for DLBCL proliferation. Mutation of MEF2B resulted in enhanced transcriptional activity of MEF2B either through disruption of its interaction with the corepressor CABIN1 or by rendering it insensitive to inhibitory signaling events mediated by phosphorylation and sumoylation. Consequently, the transcriptional activity of Bcl-6 was deregulated in DLBCLs with MEF2B mutations. Thus, somatic mutations of MEF2B may contribute to lymphomagenesis by deregulating BCL6 expression, and MEF2B may represent an alternative target for blocking Bcl-6 activity in DLBCLs.
Assuntos
Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Proteínas de Domínio MADS/genética , Mutação , Fatores de Regulação Miogênica/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Ciclo Celular/genética , Proliferação de Células , Análise por Conglomerados , Biologia Computacional , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Perfilação da Expressão Gênica , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas de Domínio MADS/química , Proteínas de Domínio MADS/metabolismo , Fatores de Transcrição MEF2 , Camundongos , Simulação de Acoplamento Molecular , Fatores de Regulação Miogênica/química , Fatores de Regulação Miogênica/metabolismo , Ligação Proteica , Conformação Proteica , Proto-Oncogene Mas , Sumoilação/genética , Transcrição GênicaRESUMO
Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement (ADE) in vitro and extend their half-lives. Here we report on prophylactic coadministration of the Fc-modified antibodies to pregnant rhesus macaques challenged three times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission, protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Complicações Infecciosas na Gravidez/prevenção & controle , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Feto/imunologia , Feto/virologia , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/administração & dosagem , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/virologia , Engenharia de Proteínas , RNA Viral/isolamento & purificação , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Zika virus/genética , Zika virus/patogenicidade , Infecção por Zika virus/imunologia , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologiaRESUMO
Higher order compaction of the eukaryotic genome is key to the regulation of all DNA-templated processes, including transcription. This tightly controlled process involves the formation of mononucleosomes, the fundamental unit of chromatin, packaged into higher order architectures in an H1 linker histone-dependent process. While much work has been done to delineate the precise mechanism of this event in vitro and in vivo, major gaps still exist, primarily due to a lack of molecular tools. Specifically, there has never been a successful purification and biochemical characterization of all human H1 variants. Here we present a robust method to purify H1 and illustrate its utility in the purification of all somatic variants and one germline variant. In addition, we performed a first ever side-by-side biochemical comparison, which revealed a gradient of nucleosome binding affinities and compaction capabilities. These data provide new insight into H1 redundancy and lay the groundwork for the mechanistic investigation of disease-driving mutations.
Assuntos
Histonas/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas Recombinantes/isolamento & purificação , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Nuclease do Micrococo/metabolismo , Nucleossomos/metabolismo , Biblioteca de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína SUMO-1/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is an increasingly diagnosed cancer that kills 90% of afflicted patients, with most patients receiving palliative chemotherapy. We identified neuronal pentraxin 1 (NPTX1) as a cancer secreted protein that becomes over-expressed in human and murine PDAC cells during metastatic progression and identified adhesion molecule with Ig like domain 2 (AMIGO2) as its receptor. Molecular, genetic, biochemical and pharmacologic experiments revealed that secreted NPTX1 acts cell-autonomously on the AMIGO2 receptor to drive PDAC metastatic colonization of the liver-the primary site of PDAC metastasis. NPTX1-AMIGO2 signaling enhanced hypoxic growth and was critically required for hypoxia induced factor-1a (HIF1a) nuclear retention and function. NPTX1 is over-expressed in human PDAC tumors and upregulated in liver metastases. Therapeutic targeting of NPTX1 with a high-affinity monoclonal antibody substantially reduced PDAC liver metastatic colonization. We thus identify NPTX1-AMIGO2 as druggable critical upstream regulators of the HIF1a hypoxic response in PDAC.
RESUMO
BACKGROUND: CD33 is a tractable target in acute myeloid leukemia (AML) for chimeric antigen receptor (CAR) T cell therapy, but clinical success is lacking. METHODS: We developed 3P14HLh28Z, a novel CD33-directed CD28/CD3Z-based CAR T cell derived from a high-affinity binder obtained through membrane-proximal fragment immunization in humanized mice. RESULTS: We found that immunization exclusively with the membrane-proximal domain of CD33 is necessary for identification of membrane-proximal binders in humanized mice. Compared with clinically validated lintuzumab-based CAR T cells targeting distal CD33 epitopes, 3P14HLh28Z showed enhanced in vitro functionality as well as superior tumor control and increased overall survival in both low antigen density and clinically relevant patient-derived xenograft models. Increased activation and enhanced polyfunctionality led to enhanced efficacy. CONCLUSIONS: Showing for the first time that a membrane-proximal CAR is superior to a membrane-distal one in the setting of CD33 targeting, our results demonstrate the rationale for targeting membrane-proximal epitopes with high-affinity binders. We also demonstrate the importance of optimizing CAR T cells for functionality in settings of both low antigen density and clinically relevant patient-derived models.
Assuntos
Imunoterapia Adotiva , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Humanos , Animais , Camundongos , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular TumoralRESUMO
Activation of PD-1 by anchoring it to Antigen Receptor (AR) components or associated co-receptors represents an attractive approach to treat autoimmune conditions. In this study, we provide evidence that CD48, a common lipid raft and Src kinase-associated coreceptor, induces significant Src kinase-dependent activation of PD-1 upon crosslinking, while CD71, a receptor excluded from these compartments, does not. Functionally, using bead-conjugated antibodies we demonstrate that CD48-dependent activation of PD-1 inhibits proliferation of AR-induced primary human T cells, and similarly, PD-1 activation using PD-1/CD48 bispecific antibodies inhibits IL-2, enhances IL-10 secretion, and reduces NFAT activation in primary human and Jurkat T cells, respectively. As a whole, CD48-dependent activation of PD-1 represents a novel mechanism to fine tune T cell activation, and by functionally anchoring PD-1 with receptors other than AR, this study provides a conceptual framework for rational development of novel therapies that activate inhibitory checkpoint receptors for treatment of immune-mediated diseases.
Assuntos
Ativação Linfocitária , Receptor de Morte Celular Programada 1 , Humanos , Células Jurkat , Quinases da Família src , ApoptoseRESUMO
Human monoclonal antibodies (mAbs) that target the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein have been isolated from convalescent individuals and developed into therapeutics for SARS-CoV-2 infection. However, therapeutic mAbs for SARS-CoV-2 have been rendered obsolete by the emergence of mAb-resistant virus variants. Here we report the generation of a set of six human mAbs that bind the human angiotensin-converting enzyme-2 (hACE2) receptor, rather than the SARS-CoV-2 spike protein. We show that these antibodies block infection by all hACE2 binding sarbecoviruses tested, including SARS-CoV-2 ancestral, Delta and Omicron variants at concentrations of ~7-100 ng ml-1. These antibodies target an hACE2 epitope that binds to the SARS-CoV-2 spike, but they do not inhibit hACE2 enzymatic activity nor do they induce cell-surface depletion of hACE2. They have favourable pharmacology, protect hACE2 knock-in mice against SARS-CoV-2 infection and should present a high genetic barrier to the acquisition of resistance. These antibodies should be useful prophylactic and treatment agents against any current or future SARS-CoV-2 variants and might be useful to treat infection with any hACE2-binding sarbecoviruses that emerge in the future.
Assuntos
COVID-19 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Humanos , Animais , Camundongos , SARS-CoV-2 , COVID-19/prevenção & controle , Anticorpos Monoclonais/farmacologiaRESUMO
Hepatitis C virus is a major global health problem affecting an estimated 170 million people worldwide. Chronic infection is common and can lead to cirrhosis and liver cancer. There is no vaccine available and current therapies have met with limited success. The viral RNA genome encodes a polyprotein that includes two proteases essential for virus replication. The NS2-3 protease mediates a single cleavage at the NS2/NS3 junction, whereas the NS3-4A protease cleaves at four downstream sites in the polyprotein. NS3-4A is characterized as a serine protease with a chymotrypsin-like fold, but the enzymatic mechanism of the NS2-3 protease remains unresolved. Here we report the crystal structure of the catalytic domain of the NS2-3 protease at 2.3 A resolution. The structure reveals a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer, and the nucleophilic cysteine by the other. The carboxy-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. Proteolysis through formation of a composite active site occurs in the context of the viral polyprotein expressed in mammalian cells. These features offer unexpected insights into polyprotein processing by hepatitis C virus and new opportunities for antiviral drug design.
Assuntos
Domínio Catalítico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Hepacivirus/enzimologia , Animais , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Cristalização , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Conformação Proteica , Eletricidade EstáticaRESUMO
More effective treatments are needed for human papilloma virus (HPV)-induced cancers despite HPV virus vaccination. The oncogenic HPV protein targets are currently undruggable and intracellular and therefore there are no antibodies to these targets. Here we report the discovery of TCR mimic monoclonal antibodies (TCRm mAb) specific for the HPV E7 protein p11-19, YMLDLQPET, when presented on the cell surface in the context of HLA-A*02:01 by use of human phage display libraries. One of the mAbs, 3F8, was able to specifically mediate T cell- redirected cytotoxicity, in a bispecific T cell engager (BiTE) form. While further studies are required to assess the therapeutic potential of this approach, the study provided the proof of concept that TCRm mAb could be a therapeutic strategy for HPV-induced human cancers.
Assuntos
Antineoplásicos Imunológicos , Neoplasias , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Anticorpos Monoclonais , Epitopos , Antígenos HLA-A , Papillomavirus Humano 16 , Humanos , Neoplasias/tratamento farmacológico , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/tratamento farmacológico , Receptores de Antígenos de Linfócitos TRESUMO
Upregulation of inhibitory receptors, such as lymphocyte activation gene-3 (LAG-3), may limit the antitumor activity of therapeutic antibodies targeting the programmed cell death protein-1 (PD-1) pathway. We describe the binding properties of ezabenlimab, an anti-human PD-1 antibody, and BI 754111, an anti-human LAG-3 antibody, and assess their activity alone and in combination. Ezabenlimab bound with high affinity to human PD-1 (KD = 6 nM) and blocked the interaction of PD-1 with PD-L1 and PD-L2. Ezabenlimab dose-dependently increased interferon-γ secretion in human T cells expressing PD-1 in co-culture with PD-L1-expressing dendritic cells. Administration of ezabenlimab to human PD-1 knock-in mice dose-dependently inhibited growth of MC38 tumors. To reduce immunogenicity, ezabenlimab was reformatted from a human IgG4 to a chimeric variant with a mouse IgG1 backbone (BI 905725) for further in vivo studies. Combining BI 905725 with anti-mouse LAG-3 antibodies improved antitumor activity versus BI 905725 monotherapy in the MC38 tumor model. We generated BI 754111, which bound with high affinity to human LAG-3 and prevented LAG-3 interaction with its ligand, major histocompatibility complex class II. In an in vitro model of antigen-experienced memory T cells expressing PD-1 and LAG-3, interferon-γ secretion increased by an average 1.8-fold versus isotype control (p = 0.027) with BI 754111 monotherapy, 6.9-fold (p < 0.0001) with ezabenlimab monotherapy and 13.2-fold (p < 0.0001) with BI 754111 plus ezabenlimab. Overall, ezabenlimab and BI 754111 bound to their respective targets with high affinity and prevented ligand binding. Combining ezabenlimab with BI 754111 enhanced in vitro activity versus monotherapy, supporting clinical investigation of this combination (NCT03156114; NCT03433898).
Assuntos
Antígeno B7-H1 , Receptor de Morte Celular Programada 1 , Animais , Anticorpos Bloqueadores , Anticorpos Monoclonais/farmacologia , Estudos Clínicos como Assunto , Inibidores de Checkpoint Imunológico , Interferon gama , Ligantes , CamundongosRESUMO
Phosphopeptides derived from dysregulated protein phosphorylation in cancer cells can be processed and presented by MHC class I and class II molecules and, therefore, represent an untapped class of tumor-specific antigens that could be used as widely expressed "public" cancer neoantigens (NeoAgs). We generated a TCR mimic (TCRm) mAb, 6B1, specific for a phosphopeptide derived from insulin receptor substrate 2 (pIRS2) presented by HLA-A*02:01. The pIRS2 epitope's presentation by HLA-A*02:01 was confirmed by mass spectrometry. The TCRm 6B1 specifically bound to pIRS2/HLA-A2 complex on tumor cell lines that expressed pIRS2 in the context of HLA-A*02:01. Bispecific mAbs engaging CD3 of T cells were able to kill tumor cell lines in a pIRS2- and HLA-A*02:01-restricted manner. Structure modeling shows a prerequisite for an arginine or lysine at the first position to bind mAb. Therefore, 6B1 could recognize phosphopeptides derived from various phosphorylated proteins with similar amino acid compositions. This raised the possibility that a TCRm specific for the pIRS2/HLA-A2 complex could target a range of phosphopeptides presented by HLA-A*02:01 in various tumor cells. This is the first TCRm mAb to our knowledge targeting a phosphopeptide/MHC class I complex; the potential of this class of agents for clinical applications warrants further investigation.
Assuntos
Antígeno HLA-A2 , Fosfopeptídeos , Anticorpos Monoclonais/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Fosfopeptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Most patients with non-small cell lung cancer (NSCLC) do not achieve durable clinical responses from immune checkpoint inhibitors, suggesting the existence of additional resistance mechanisms. Nicotinamide adenine dinucleotide (NAD)-induced cell death (NICD) of P2X7 receptor (P2X7R)-expressing T cells regulates immune homeostasis in inflamed tissues. This process is mediated by mono-adenosine 5'-diphosphate (ADP)-ribosyltransferases (ARTs). We found an association between membranous expression of ART1 on tumor cells and reduced CD8 T cell infiltration. Specifically, we observed a reduction in the P2X7R+ CD8 T cell subset in human lung adenocarcinomas. In vitro, P2X7R+ CD8 T cells were susceptible to ART1-mediated ADP-ribosylation and NICD, which was exacerbated upon blockade of the NAD+-degrading ADP-ribosyl cyclase CD38. Last, in murine NSCLC and melanoma models, we demonstrate that genetic and antibody-mediated ART1 inhibition slowed tumor growth in a CD8 T cell-dependent manner. This was associated with increased infiltration of activated P2X7R+CD8 T cells into tumors. In conclusion, we describe ART1-mediated NICD as a mechanism of immune resistance in NSCLC and provide preclinical evidence that antibody-mediated targeting of ART1 can improve tumor control, supporting pursuit of this approach in clinical studies.
Assuntos
ADP Ribose Transferases , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Subpopulações de Linfócitos T , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Difosfato de Adenosina , Animais , Carcinoma Pulmonar de Células não Pequenas/imunologia , Proteínas Ligadas por GPI/genética , Humanos , Neoplasias Pulmonares/imunologia , CamundongosRESUMO
Hedgehog signaling is essential for bone formation, including functioning as a means for the growth plate to drive skeletal mineralization. However, the mechanisms regulating hedgehog signaling specifically in bone-forming osteoblasts are largely unknown. Here, we identified SLIT and NTRK-like protein-5(Slitrk5), a transmembrane protein with few identified functions, as a negative regulator of hedgehog signaling in osteoblasts. Slitrk5 is selectively expressed in osteoblasts and loss of Slitrk5 enhanced osteoblast differentiation in vitro and in vivo. Loss of SLITRK5 in vitro leads to increased hedgehog signaling and overexpression of SLITRK5 in osteoblasts inhibits the induction of targets downstream of hedgehog signaling. Mechanistically, SLITRK5 binds to hedgehog ligands via its extracellular domain and interacts with PTCH1 via its intracellular domain. SLITRK5 is present in the primary cilium, and loss of SLITRK5 enhances SMO ciliary enrichment upon SHH stimulation. Thus, SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts that may be attractive as a therapeutic target to enhance bone formation.
Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Receptor Patched-1/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Proteínas Hedgehog/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Osteoblastos/citologia , Receptor Patched-1/genética , Transdução de SinaisRESUMO
The hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a dimeric multifunctional hydrophobic protein with an essential but poorly understood role in infectious virus production. We investigated the determinants of NS2 function in the HCV life cycle. On the basis of the crystal structure of the postcleavage form of the NS2 protease domain, we mutated conserved features and analyzed the effects of these changes on polyprotein processing, replication, and infectious virus production. We found that mutations around the protease active site inhibit viral RNA replication, likely by preventing NS2-3 cleavage. In contrast, alterations at the dimer interface or in the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity but decreased infectious virus production. A comprehensive deletion and mutagenesis analysis of the C-terminal end of NS2 revealed the importance of its C-terminal leucine residue in infectious particle production. The crystal structure of the NS2 protease domain shows that this C-terminal leucine is locked in the active site, and mutation or deletion of this residue could therefore alter the conformation of NS2 and disrupt potential protein-protein interactions important for infectious particle production. These studies begin to dissect the residues of NS2 involved in its multiple essential roles in the HCV life cycle and suggest NS2 as a viable target for HCV-specific inhibitors.
Assuntos
Hepacivirus/fisiologia , Proteínas não Estruturais Virais/química , Células Cultivadas , Dimerização , Epitopos , Genoma Viral , Hepacivirus/genética , Humanos , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/fisiologia , Replicação ViralRESUMO
The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.
Assuntos
Proteínas de Transporte/metabolismo , Hepacivirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/fisiologia , Proteínas Virais/metabolismo , Montagem de Vírus , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Proteínas de Transporte/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Ligação Proteica , Mapeamento de Interação de Proteínas , Supressão Genética , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genéticaRESUMO
Hepatitis C virus (HCV) p7 is an integral membrane protein that forms ion channels in vitro and that is crucial for the efficient assembly and release of infectious virions. Due to these properties, p7 was included in the family of viroporins that comprises proteins like influenza A virus M2 and human immunodeficiency virus type 1 (HIV-1) vpu, which alter membrane permeability and facilitate the release of infectious viruses. p7 from different HCV isolates sustains virus production with variable efficiency. Moreover, p7 determinants modulate processing at the E2/p7 and the p7/NS2 signal peptidase cleavage sites, and E2/p7 cleavage is incomplete. Consequently, it was unclear if a differential ability to sustain virus production was due to variable ion channel activity or due to alternate processing at these sites. Therefore, we developed a trans-complementation assay permitting the analysis of p7 outside of the HCV polyprotein and thus independently of processing. The rescue of p7-defective HCV genomes was accomplished by providing E2, p7, and NS2, or, in some cases, by p7 alone both in a transient complementation assay as well as in stable cell lines. In contrast, neither influenza A virus M2 nor HIV-1 vpu compensated for defective p7 in HCV morphogenesis. Thus, p7 is absolutely essential for the production of infectious HCV particles. Moreover, our data indicate that p7 can operate independently of an upstream signal sequence, and that a tyrosine residue close to the conserved dibasic motif of p7 is important for optimal virus production in the context of genotype 2a viruses. The experimental system described here should be helpful to investigate further key determinants of p7 that are essential for its structure and function in the absence of secondary effects caused by altered polyprotein processing.
Assuntos
Hepacivirus/fisiologia , Proteínas Virais/fisiologia , Montagem de Vírus/fisiologia , Northern Blotting , Western Blotting , Linhagem Celular , Teste de Complementação Genética , Humanos , Replicação Viral/fisiologiaRESUMO
Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.
Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/genética , Células Dendríticas/imunologia , RNA Mensageiro/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Bromovirus/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Cricetinae , Células Dendríticas/virologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Camundongos , RNA Mensageiro/imunologia , Análise de Célula Única , Montagem de VírusRESUMO
The structure of the HIV-1 envelope membrane-proximal external region (MPER) is influenced by its association with the lipid bilayer on the surface of virus particles and infected cells. To develop a replicating vaccine vector displaying MPER sequences in association with membrane, Env epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, or both were grafted into the membrane-proximal stem region of the vesicular stomatitis virus (VSV) glycoprotein (G). VSV encoding functional G-MPER chimeras based on G from the Indiana or New Jersey serotype propagated efficiently, although grafting of both epitopes (G-2F5-4E10) modestly reduced replication and resulted in the acquisition of one to two adaptive mutations in the grafted MPER sequence. Monoclonal antibodies 2F5 and 4E10 efficiently neutralized VSV G-MPER vectors and bound to virus particles in solution, indicating that the epitopes were accessible in the preattachment form of the G-MPER chimeras. Overall, our results showed that the HIV Env MPER could functionally substitute for the VSV G-stem region implying that both perform similar functions even though they are from unrelated viruses. Furthermore, we found that the MPER sequence grafts induced low but detectable MPER-specific antibody responses in rabbits vaccinated with live VSV, although additional vector and immunogen modifications or use of a heterologous prime-boost vaccination regimen will be required to increase the magnitude of the immune response.
Assuntos
Anticorpos Anti-HIV/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Vesiculovirus/fisiologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Feminino , Glicoproteínas de Membrana/imunologia , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vesiculovirus/genética , Vesiculovirus/crescimento & desenvolvimento , Vesiculovirus/imunologia , Proteínas do Envelope Viral/imunologia , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection.