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1.
Vet Microbiol ; 239: 108462, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767100

RESUMO

In contrast to human influenza viruses that replicate in the respiratory tract and are airborne transmitted, avian viruses also replicate in gut epithelial cells and are transmitted via the fecal-oral route. On this route, the virus is exposed to destructive fluids of the digestive tract, which are acidic and contain the proteases pepsin (gizzard) or chymotrypsin and trypsin (intestine). Only the latter enzyme activates virus by cleaving hemagglutinin (HA) into HA1 and HA2 subunits. We mimicked the passage of viruses through the gastrointestinal tract by treating them with digestive fluids from chicken and determined titers and integrity of HA by western-blot. Gizzard fluid completely inactivated virions and degrades HA even at a high dilution, but only if the pH was kept acidic. If the fluid is diluted with neutral buffer (mimicking virus uptake with seawater) particles were more resistant. Virions containing an uncleaved HA were even activated suggesting that gastric juice contains a trypsin-like protease. Undiluted intestinal fluid inactivated particles and destroyed HA, but diluted fluid activated virions. A virus isolated from the duck´s intestine is more tolerant against intestinal fluid compared to fowl plague virus suggesting that the former is better adapted to grow in the intestine. We also demonstrate that influenza viruses replicate to high titers in a novel chicken epithelial gut cell line. While viruses with a monobasic HA cleavage site require addition of trypsin, these cells effectively process HA with a polybasic cleavage site, which could be blocked with an inhibitor of the cellular furin protease.


Assuntos
Trato Gastrointestinal/virologia , Hemaglutininas/metabolismo , Influenza Aviária/virologia , Animais , Galinhas , Células Epiteliais/citologia , Células Epiteliais/virologia , Suco Gástrico/química , Suco Gástrico/enzimologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Secreções Intestinais/química , Secreções Intestinais/enzimologia , Inativação de Vírus , Replicação Viral/fisiologia
2.
Chem Pharm Bull (Tokyo) ; 67(11): 1201-1207, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31434835

RESUMO

Oleanolic acid (OA) was discovered as a mild influenza hemagglutinin (HA) inhibitor in our earlier studies. In the present work, 20 compounds were prepared by structural modifications of OA, and their antiviral activities against influenza A/WSN/33 (H1N1) virus in Madin-Darby canine kidney (MDCK) cells were evaluated. Based on the biological result, structure-activity relationship (SAR) was discussed. Compound 10 with six-carbon chain and a terminal hydroxyl group showed the strongest anti-influenza activity with an IC50 of 2.98 µM, which is an order of magnitude more potent than OA. Hemagglutination inhibition and Surface plasmon resonance (SPR) assay indicated that compound 10 might interfere with influenza invasion by interacting with HA protein.


Assuntos
Antivirais/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Ácido Oleanólico/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Cães , Relação Dose-Resposta a Droga , Hemaglutininas/efeitos dos fármacos , Hemaglutininas/metabolismo , Vírus da Influenza A/metabolismo , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Estrutura Molecular , Ácido Oleanólico/síntese química , Ácido Oleanólico/química , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
3.
Nanoscale ; 11(29): 13878-13884, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31304500

RESUMO

Stimulation of dendritic cells (DCs) by antigens (Ags) promotes an Ag-specific immune response that kills Ag-expressing pathogens. These biologically inspired nanocarriers have received much attention as tools to deliver cancer Ags to DCs. A polymer-templated protein nanoball having hemagglutinin (H1-NB) that mimics the influenza virus can be used as a cancer Ag delivery vehicle, as DCs show effective phagocytic activities against H1-NB without any adjuvant. In the present study, H1-NB containing ovalbumin (OVA), a model Ag (H1-OVA-NB), was prepared as an anti-cancer agent and evaluated for its effect on anticancer immunity. H1-OVA-NB treatments in C57BL/6 mice enhanced OVA-specific immune activation and efficiently inhibited B16-OVA tumor growth compared to control groups. Our results indicate that H1-NB is an effective carrier for Ag delivery to DCs and promotes immunotherapy to fight cancer.


Assuntos
Antígenos/imunologia , Células Dendríticas/imunologia , Hemaglutininas/química , Imunoterapia , Nanopartículas/química , Polímeros/química , Animais , Antígenos/química , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Portadores de Fármacos/química , Hemaglutininas/genética , Hemaglutininas/metabolismo , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Orthomyxoviridae/metabolismo , Ovalbumina/genética , Ovalbumina/imunologia , Ovalbumina/metabolismo
4.
BMC Res Notes ; 12(1): 358, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234908

RESUMO

OBJECTIVE: Urinary tract infection (UTI) is one of the most frequent disease encounters in pregnant mothers, and the most drug resistant, biofilm and hemagglutinin producer Uropathogenic Escherichia coli (UPEC) is the major etiologic agent. Therefore, the aim of this study was to assess the association between the antimicrobial resistance, and biofilm and hemagglutinin production of Uropathogenic Escherichia coli. RESULTS: UTI among the study participants was 27.3%; and UPEC was found the major etiologic agent followed by coagulase negative staphylococcus. Risk factors, previous history of catheterization and previous history of UTI were found significantly associated with UTI, recurrent UTI, drug resistance and biofilm formation. Of the tested antibiotics, nitrofurantoin was the most effective drug for UPEC. Nearly 100% of the biofilm producers were resistant to norfloxacin, cotrimoxazole, and gentamicin.


Assuntos
Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana , Hemaglutininas/metabolismo , Escherichia coli Uropatogênica/fisiologia , Adolescente , Adulto , Feminino , Humanos , Modelos Logísticos , Análise Multivariada , Adulto Jovem
5.
Antonie Van Leeuwenhoek ; 112(11): 1655-1662, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31230158

RESUMO

Actinobacillus seminis, a commensal of ovine and caprine reproductive organs, is able to induce epididymitis in the small ruminants that it infects. In this work, we characterised two protein bands of approximately 150 kDa and 65 kDa. These proteins cross-reacted with a polyclonal serum against Gallibacterium anatis hemagglutinin and with a polyclonal serum from sheep with epididymitis, indicating that the proteins are expressed in vivo; the two proteins also interacted with biotin-labeled sheep fibrinogen and fibronectin, suggesting that they may function as adhesins. The participation of these proteins as adhesins was confirmed by a cultured human bladder cell-A. seminis adhesion assay and adherence inhibition by preincubation of A. seminis with polyclonal antiserum to the 150 kDa protein. Both proteins presented sequence identity with an A. seminis GroEL protein by mass spectrometry analysis and agglutinated glutaraldehyde-fixed sheep red blood cells. Immunogold labeling was observed by transmission electron microscopy on bacterial cells that were negatively stained, and a peroxidase reaction was detected in A. seminis biofilms, when an anti-A. seminis 150 kDa protein serum was used, indicating the presence of this protein on the surface of A. seminis and in biofilms. The A. seminis GroEL-homologue is a multifunctional protein that likely acts as a hemagglutinin.


Assuntos
Actinobacillus seminis/fisiologia , Eritrócitos/imunologia , Proteínas de Choque Térmico/imunologia , Proteínas de Choque Térmico/metabolismo , Aglutinação , Testes de Aglutinação , Animais , Anticorpos Antibacterianos , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Biofilmes , Adesão Celular , Eritrócitos/metabolismo , Proteínas de Choque Térmico/isolamento & purificação , Hemaglutinação , Hemaglutininas/metabolismo , Ovinos
6.
Vet Res ; 50(1): 37, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118100

RESUMO

The fusogenically activated F and HN proteins of virulent NDV induce complete autophagic flux in DF-1 and A549 cells. However, the effect of both glycoproteins on mitochondria remains elusive. Here, we found that F and HN cooperation increases mitochondrial biogenesis but does not cause the mitochondria damage. We observed that both glycoproteins change the morphological characteristics and spatial distribution of intracellular mitochondria. F and HN cooperate cooperatively to induce ER stress and UPRmt. Our preliminary data suggested that F and HN cooperatively disturb mitochondrial fusion-fission homeostasis to enhance mitochondrial biogenesis, and eventually meet the energy demand of syncytium formation.


Assuntos
Retículo Endoplasmático/virologia , Hemaglutininas/metabolismo , Mitocôndrias/metabolismo , Neuraminidase/metabolismo , Doença de Newcastle/metabolismo , Vírus da Doença de Newcastle/metabolismo , Resposta a Proteínas não Dobradas , Células A549/metabolismo , Células A549/virologia , Animais , Western Blotting , Retículo Endoplasmático/metabolismo , Homeostase , Humanos , Mitocôndrias/virologia
7.
Nat Commun ; 10(1): 1799, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996301

RESUMO

Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Hemaglutininas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Influenza Humana/imunologia , Oligossacarídeos/metabolismo , Animais , Proteínas de Bactérias/genética , Bioensaio/métodos , Células CHO , Cricetulus , Cães , Glicosiltransferases/genética , Voluntários Saudáveis , Helicobacter mustelae/genética , Helicobacter mustelae/metabolismo , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Microscopia Intravital/métodos , Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Pulmão/patologia , Células Madin Darby de Rim Canino , Engenharia Metabólica/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Oligossacarídeos/imunologia , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem/métodos
8.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30927348

RESUMO

Bacteria use a variety of mechanisms to translocate proteins from the cytoplasm, where they are synthesized, to the cell surface or extracellular environment or directly into other cells, where they perform their ultimate functions. Type V secretion systems (T5SS) use ß-barrel transporter domains to export passenger domains across the outer membranes of Gram-negative bacteria. Distinct among T5SS are type Vb or two-partner secretion (TPS) systems in which the transporter and passenger are separate proteins, necessitating a mechanism for passenger-translocator recognition in the periplasm and providing the potential for reuse of the translocator. This review describes current knowledge of the TPS translocation mechanism, using Bordetella filamentous hemagglutinin (FHA) and its transporter FhaC as a model. We present the hypothesis that the TPS pathway may be a general mechanism for contact-dependent delivery of toxins to target cells.


Assuntos
Bordetella/metabolismo , Hemaglutininas/metabolismo , Via Secretória/fisiologia , Adesinas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Bordetella/patogenicidade , Bordetella pertussis/metabolismo , Bordetella pertussis/patogenicidade , Bactérias Gram-Negativas , Proteínas de Membrana Transportadoras , Modelos Moleculares , Sistemas de Secreção Tipo V/metabolismo , Virulência , Fatores de Virulência de Bordetella/metabolismo , Coqueluche/microbiologia
9.
Eur J Pharm Biopharm ; 136: 259-266, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30731115

RESUMO

Microneedle arrays (MNAs) are a promising mean to administer vaccines. Without the need of highly trained personnel, MNAs can be applied to deliver vaccines into the dermis, which is well equipped to initiate potent immune responses. While vaccination using dissolving microneedle arrays has been extensively investigated, the use of solid nanoporous MNAs (npMNAs) to deliver vaccines remained largely unexplored. In this report we investigated whether npMNAs with an average pore size of 80 nm, can be used for influenza vaccination based on recombinant hemagglutinin (HA) protein of the 2009 pandemic H1N1 (pH1N1) virus. Fluorescently labeled HA loaded in the npMNAs was effectively delivered into the skin of mouse ears, as a result of a diffusion-based process. Compared to intramuscular immunization, intradermal HA vaccination of mice using npMNAs elicited high levels of HA antigen specific antibodies, with pH1N1 hemagglutination inhibition and neutralization activity. Moreover, mice vaccinated with pH1N1 HA loaded npMNAs were completely protected against a potentially lethal challenge with mouse adapted pH1N1 virus. These results illustrate that intradermal subunit vaccine immunization using npMNAs is a promising approach to facilitate effective vaccination.


Assuntos
Hemaglutininas/administração & dosagem , Vacinas contra Influenza/administração & dosagem , Influenza Humana/prevenção & controle , Microinjeções/métodos , Nanoporos , Vacinação/métodos , Animais , Cerâmica/química , Cerâmica/farmacocinética , Cães , Hemaglutininas/química , Hemaglutininas/metabolismo , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/fisiologia , Vacinas contra Influenza/química , Vacinas contra Influenza/farmacocinética , Influenza Humana/imunologia , Influenza Humana/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Microinjeções/instrumentação , Agulhas , Vacinação/instrumentação
10.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30518651

RESUMO

Hemagglutinin (HA) stalk-reactive antibodies are the basis of several current "one-shot" universal influenza vaccine efforts because they protect against a wide spectrum of influenza virus strains. The appreciated mechanism of protection by HA stalk-reactive antibodies is to inhibit HA stalk reconfiguration, blocking viral fusion and entry. This study shows that HA stalk-reactive antibodies also inhibit neuraminidase (NA) enzymatic activity, prohibiting viral egress. NA inhibition (NI) was evident for an attached substrate but not for unattached small-molecule cleavage of sialic acid. This finding suggests that the antibodies inhibit NA enzymatic activity through steric hindrance, thus limiting NA access to sialic acids when adjacent to HA on whole virions. Consistently, F(ab')2 fragments that occupied reduced area without loss of avidity or disrupted HA/NA interactions showed significantly reduced NI activity. Notably, HA stalk-binding antibodies lacking NI activity were unable to neutralize viral infection via microneutralization assays. This work suggests that NI activity is an important component of protection mediated by HA stalk-reactive antibodies.IMPORTANCE This study reports a new mechanism of protection mediated by influenza hemagglutinin stalk-reactive antibodies, i.e., inhibition of neuraminidase activity by steric hindrance, blocking access of neuraminidase to sialic acids when it abuts hemagglutinin on whole virions.


Assuntos
Hemaglutininas/imunologia , Neuraminidase/metabolismo , Orthomyxoviridae/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Proteção Cruzada , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Hemaglutininas/metabolismo , Humanos , Imunização Passiva , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/virologia , Neuraminidase/química , Neuraminidase/imunologia , Testes de Neutralização , Infecções por Orthomyxoviridae/virologia , Proteínas Virais/química
11.
Nat Commun ; 9(1): 4879, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451858

RESUMO

Kainate-type glutamate receptors play critical roles in excitatory synaptic transmission and synaptic plasticity in the brain. GluK1 and GluK2 possess fundamentally different capabilities in surface trafficking as well as synaptic targeting in hippocampal CA1 neurons. Here we find that the excitatory postsynaptic currents (EPSCs) are significantly increased by the chimeric GluK1(SPGluK2) receptor, in which the signal peptide of GluK1 is replaced with that of GluK2. Coexpression of GluK1 signal peptide completely suppresses the gain in trafficking ability of GluK1(SPGluK2), indicating that the signal peptide represses receptor trafficking in a trans manner. Furthermore, we demonstrate that the signal peptide directly interacts with the amino-terminal domain (ATD) to inhibit the synaptic and surface expression of GluK1. Thus, we have uncovered a trafficking mechanism for kainate receptors and propose that the cleaved signal peptide behaves as a ligand of GluK1, through binding with the ATD, to repress forward trafficking of the receptor.


Assuntos
Região CA1 Hipocampal/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Sinais Direcionadores de Proteínas/genética , Receptores de Ácido Caínico/metabolismo , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Sítios de Ligação , Região CA1 Hipocampal/citologia , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hemaglutininas/genética , Hemaglutininas/metabolismo , Humanos , Microtomia , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Plasticidade Neuronal , Técnicas de Cultura de Órgãos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/genética , Sinapses/metabolismo , Sinapses/ultraestrutura
12.
Life Sci ; 213: 158-165, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30352241

RESUMO

Aim To investigate the immunomodulatory potential of a chimera composed of the receptor-binding domain of hemagglutinin 1 (H1s) from Influenza virus and Der p 2 (D2) allergen for allergen-specific immunotherapy of house-dust mite allergy (HDM). MAIN METHODS: H1sD2 chimera and D2 allergen were produced by genetic engineering in E. coli. Recombinant antigens were extracted from inclusion bodies by urea, then refolded and purified by immobilized- metal affinity chromatography (IMAC). Purity was verified by 2D-PAGE and secondary structures were assessed by CD spectroscopy. IgE reactivity of H1sD2 and D2 was tested in western blot with sera from 8 persons with clinical history of HDM allergy. Immunogenicity of H1sD2 and D2 were analyzed in Balb/c mice. Cytokine profile was analyzed by ELISA after stimulation of mouse spleen cells with H1sD2 and D2. Leukocyte population abundance of cells isolated from spleen and lymph node was assessed by flow cytometry. KEY FINDINGS: Purified recombinant proteins H1sD2 (42 kDa) and D2 (15 kDa) revealed well defined secondary structures, and preserved IgE reactive epitopes. Analysis of supernatants of mouse spleen cells after stimulation with H1sD2 and D2, revealed a qualitatively different cytokine profile from H1sD2 immunized mouse cells (increase in IL10). CD8+ cells were decreased in the lymph node of D2 immunized mice, whereas H1sD2 immunization led to an increase of CD8+ cells in both the lymph node and the spleen. SIGNIFICANCE: H1sD2 chimera attenuates Der p 2-inherent Th2 response and directs the immune response toward Th1 and Treg phenotype.


Assuntos
Alérgenos/uso terapêutico , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/metabolismo , Hemaglutininas/metabolismo , Alérgenos/metabolismo , Animais , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Quimera , Epitopos/metabolismo , Escherichia coli/imunologia , Engenharia Genética/métodos , Hemaglutininas/genética , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Orthomyxoviridae/metabolismo , Pyroglyphidae/imunologia , Proteínas Recombinantes/genética
13.
Biomaterials ; 183: 234-242, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30176403

RESUMO

The generation of virus-mimetic nanoparticles has received much attention in developing a new vaccine for overcoming the limitations of current vaccines. Thus, a method, encompassing most viral features for their size, hydrophobic domain and antigen display, would represent a meaningful direction for the vaccine development. In the present study, a polymer-templated protein nanoball with direction oriented hemagglutinin1 on its surface (H1-NB) was prepared as a new influenza vaccine, exhibiting most of the viral features. Moreover, the concentrations of antigen on the particle surface were controlled, and its effect on immunogenicity was estimated by in vivo studies. Finally, H1-NB efficiently promoted H1-specific immune activation and cross-protective activities, which consequently prevented H1N1 infections in mice.


Assuntos
Hemaglutininas Virais/metabolismo , Hemaglutininas/química , Vacinas contra Influenza/química , Nanopartículas/química , Polímeros/química , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Células Dendríticas/fisiologia , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Tamanho da Partícula , Baço/citologia
14.
Virology ; 525: 106-116, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30253275

RESUMO

In this study, we investigated the feasibility of using enterovirus HY12 as a vector to express an exogenous hemagglutinin (HA)-epitope tag onto the HY12-encoded VP1 protein via a reverse genetic system. Characteristics of recombinant (r) HY12-VP1-HA marker virus were determined by immunoperoxidase monolayer assay, western blot, electron microscopy, and serum-neutralisation assay. Sequence analysis demonstrated that the marker virus stably maintained the HA-epitope-tag in MDBK cells, with no changes in viral morphological features observed relative to those of the parental rHY12 virus. Furthermore, detection by immunofluorescence assay revealed the expression of HA-epitope tag and VP2 protein, which distinguish the marker virus from parental rHY12 virus. In addition, neonatal mice infected with the recombinant marker virus showed various microscopic pathological lesions and generated anti-HY12 virus and -HA-epitope-tag antibodies. These results indicated that the recombinant marker virus represented a valuable platform to promote the development of novel genetic vaccines.


Assuntos
Proteínas do Capsídeo/metabolismo , Enterovirus/metabolismo , Epitopos/metabolismo , Hemaglutininas/metabolismo , Animais , Animais Recém-Nascidos , Antígenos Virais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Bovinos , Linhagem Celular , Regulação Viral da Expressão Gênica , Camundongos , Testes de Neutralização , RNA Viral/genética , Distribuição Aleatória , Vírus Reordenados
15.
Vaccine ; 36(41): 6144-6151, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30194004

RESUMO

Influenza vaccines are the most effective intervention to prevent the substantial public health burden of seasonal and pandemic influenza. Hemagglutinin (HA), as the main antigen in inactivated influenza vaccines (IIVs), elicits functional neutralizing antibodies and largely determines IIV effectiveness. HA potency has been evaluated by single-radial immunodiffusion (SRID), the standard in vitro potency assay for IIVs, to predict vaccine immunogenicity with a correlation to protective efficacy. We previously reported that limited trypsin digestion (LTD) selectively degraded stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique could specifically quantify trypsin-resistant, immunologically active HA. Here, we demonstrate that isotope dilution mass spectrometry (IDMS), a method capable of quantifying the absolute HA concentration without reference antigen use, can be further expanded by adding LTD followed with precipitation to selectively quantify the active HA. We test the LTD-IDMS assay on H7N9 vaccines stressed by low pH, raised temperature, or freeze/thaw cycles. This method, unlike SRID, has no requirement for strain-specific reference antigens or antibodies and can generate potency values that correlate with SRID. Thus, LTD-IDMS is a promising alternative in vitro potency assay for influenza vaccines to complement and potentially replace SRID in a pandemic when strain specific reagents may not be readily available.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/imunologia , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/uso terapêutico , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Hemaglutininas/metabolismo , Humanos , Marcação por Isótopo , Espectrometria de Massas
16.
Artigo em Inglês | MEDLINE | ID: mdl-30150460

RESUMO

We evaluated MEDI8852, a human IgG1 monoclonal antibody that binds a highly conserved influenza A hemagglutinin stalk epitope, in outpatients with uncomplicated influenza A infection. A total of 126 subjects aged 18 to 65 years were enrolled during the 2015 to 2016 Northern and 2016 Southern Hemisphere seasons. Subjects with symptom onset ≤5 days before dosing were randomized to four cohorts: 750 mg (cohort 1) or 3,000 mg (cohort 2) MEDI8852 (single intravenous infusion) plus 75 mg oseltamivir, placebo plus 75 mg oseltamivir (cohort 3), and 3,000 mg MEDI8852 alone (cohort 4). Subjects were monitored through day 10 for solicited influenza symptoms, day 28 for adverse events (AEs), and day 101 for serious AEs and AEs of special interest. Nasopharyngeal samples were collected through day 7 for confirmation of influenza A infection, viral shedding, and oseltamivir and MEDI8852 susceptibility. Slightly more AEs were reported in subjects receiving MEDI8852 (cohorts 1, 2, and 4 combined: 39/93, 41.9%) than oseltamivir only (cohort 3: 10/32, 31.3%). Most AEs were mild or moderate. The most common AE was bronchitis (11/93, 11.8%; 1/32, 3.1%). The median (range) decrease in viral shedding (log10 virus genome copies/ml) was similar between the two groups (-3.58 [-6.2. 0.5]; -3.43 [-5.9, 0.9]). Genotypic analyses found a limited number of hemagglutinin and neuraminidase amino acid changes between viruses isolated before and after therapy; however, none appeared within a known oseltamivir-resistant site or MEDI8852-binding region. The safety profile of MEDI8852 supports its continued development for treatment of patients hospitalized with influenza A infection. (This study has been registered at ClinicalTrials.gov under identifier NCT02603952.).


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antivirais/uso terapêutico , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Adolescente , Adulto , Idoso , Método Duplo-Cego , Feminino , Hemaglutininas/metabolismo , Humanos , Influenza Humana/metabolismo , Masculino , Pessoa de Meia-Idade , Neuraminidase/metabolismo , Oseltamivir/uso terapêutico , Eliminação de Partículas Virais/efeitos dos fármacos , Adulto Jovem
17.
Biochemistry ; 57(37): 5480-5493, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30141905

RESUMO

Cellular entry of influenza virus is mediated by the viral protein hemagglutinin (HA), which forms an initial complex of three HA1 and three HA2 subunits. Each HA2 includes a fusion peptide (FP), a soluble ectodomain (SE), and a transmembrane domain. HA1 binds to cellular sialic acids, followed by virus endocytosis, pH reduction, dissociation of HA1, and structural rearrangement of HA2 into a final trimer-of-SE hairpins. A decrease in pH also triggers HA2-mediated virus/endosome membrane fusion. SE hairpins have an interior parallel helical bundle and C-terminal strands in the grooves of the exterior of the bundle. FPs are separate helical hairpins. This study compares wild-type HA2 (WT-HA2) with G1E(FP) and I173E(SE strand) mutants. WT-HA2 induces vesicle fusion at pH 5.0, whereas the extent of fusion is greatly reduced for both mutants. Circular dichroism for HA2 and FHA2≡FP+SE constructs shows dramatic losses of stability for the mutants, including a Tm reduced by 40 °C for I173E-FHA2. This is evidence of destabilization of SE hairpins via dissociation of strands from the helical bundle, which is also supported by larger monomer fractions for mutant versus WT proteins. The G1E mutant may have disrupted FP hairpins, with consequent non-native FP binding to dissociated SE strands. It is commonly proposed that free energy released by the HA2 structural rearrangement catalyzes HA-mediated fusion. This study supports an alternate mechanistic model in which fusion is preceded by FP insertion in the target membrane and formation of the final SE hairpin. Less fusion by the mutants is due to the loss of hairpin stability and consequent reduced level of membrane apposition of the virus and target membranes.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Hemaglutininas Virais/química , Hemaglutininas/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Hemaglutininas Virais/metabolismo , Humanos , Conformação Proteica , Domínios Proteicos , Subunidades Proteicas
18.
J Gen Virol ; 99(9): 1187-1198, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30084768

RESUMO

The haemagglutinin (HA) of H1N1 and H3N2 influenza A virus (IAV) subtypes has to be activated by host proteases. Previous studies showed that H1N1 virus cannot replicate efficiently in Tmprss2-/- knock-out mice whereas H3N2 viruses are able to replicate to the same levels in Tmprss2-/- as in wild type (WT) mice. Here, we investigated the sequence requirements for the HA molecule that allow IAV to replicate efficiently in the absence of TMPRSS2. We showed that replacement of the H3 for the H1-loop sequence (amino acids 320 to 329, at the C-terminus of HA1) was not sufficient for equal levels of virus replication or severe pathology in Tmprss2-/- knock-out mice compared to WT mice. However, exchange of a distant amino acid from H1 to H3 sequence (E31D) in addition to the HA-loop substitution resulted in virus replication in Tmprss2-/- knock-out mice that was comparable to WT mice. The higher virus replication and lung damage was associated with increased epithelial damage and higher mortality. Our results provide further evidence and insights into host proteases as a promising target for therapeutic intervention of IAV infections.


Assuntos
Hemaglutininas/metabolismo , Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/virologia , Serina Endopeptidases/metabolismo , Replicação Viral/fisiologia , Substituição de Aminoácidos , Animais , Clonagem Molecular , Cães , Regulação Viral da Expressão Gênica/fisiologia , Hemaglutininas/química , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Modelos Moleculares , Mutagênese , Conformação Proteica , Serina Endopeptidases/genética
19.
Int J Biol Macromol ; 119: 533-539, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30071221

RESUMO

Ovomucin (OVM) plays an important role in inhibiting infection of various pathogens. However, this bioactivity mechanism is not much known. Here, the role of sialic acid in OVM anti-virus activity has been studied by ELISA with lectin or ligand. Structural changes of OVM after removing sialic acid were analyzed by circular dichroism and fluorescence spectroscopy. OVM could be binding to the hemagglutinin (HA) of avian influenza viruses H5N1 and H1N1, this binding was specific and required the involvement of sialic acid. When sialic acid was removed, the binding was significantly reduced 71.5% and 64.35%, respectively. Therefore, sialic acid was proved as a recognition site which avian influenza virus bound to. Meanwhile, the endogenous fluorescence and surface hydrophobicity of OVM removing sialic acid were increased and the secondary structure tended to shift to random coil. This indicated that OVM molecules were in an unfolded state and spatial conformation disorder raising weakly. Remarkably, free sialic acid strongly promoted OVM binding to HA and thereby enhanced the interaction. It may contribute to the inhibition of host cell infection, agglutinate viruses. This study can be extended to the deepening of passive immunization field.


Assuntos
Antivirais/química , Antivirais/metabolismo , Hemaglutininas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Ovomucina/química , Ovomucina/metabolismo , Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/metabolismo , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/metabolismo , Ovomucina/farmacologia
20.
PLoS Biol ; 16(7): e2006128, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30005062

RESUMO

The mitochondrial F-type ATP synthase, a multisubunit nanomotor, is critical for maintaining cellular ATP levels. In T. gondii and other apicomplexan parasites, many subunit components necessary for proper assembly and functioning of this enzyme appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomeric (approximately 600 kDa) and dimeric (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits a, b, and d can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid, and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex could facilitate the development of novel antiparasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.


Assuntos
ATPases Mitocondriais Próton-Translocadoras/metabolismo , Subunidades Proteicas/metabolismo , Toxoplasma/enzimologia , Sequência de Aminoácidos , Animais , Sequência Conservada , Regulação da Expressão Gênica , Variação Genética , Hemaglutininas/metabolismo , Mitocôndrias/metabolismo , Parasitos/metabolismo , Filogenia , Plasmodium falciparum/metabolismo , Multimerização Proteica , Proteoma/metabolismo , Proteômica , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
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