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1.
Science ; 368(6492)2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32409444

RESUMO

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo-designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.


Assuntos
Anticorpos Neutralizantes/biossíntese , Biologia Computacional/métodos , Epitopos Imunodominantes/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Vacinas contra Vírus Sincicial Respiratório/química , Vírus Sincicial Respiratório Humano/imunologia , Motivos de Aminoácidos , Humanos , Epitopos Imunodominantes/imunologia , Conformação Proteica , Proteínas Recombinantes de Fusão/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia
2.
PLoS One ; 15(3): e0229660, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32191728

RESUMO

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract disease in infants and young children worldwide and a high priority for vaccine development. Despite over 50 years of research, however, no vaccine is yet available. One block to vaccine development is an incomplete understanding of the aberrant memory response to the formalin-inactivated RSV vaccine (FI-RSV) given to children in the 1960s. This vaccine caused enhanced respiratory disease (ERD) with later natural RSV infection. Concern that any non-live virus vaccine may also cause ERD has blocked development of subunit vaccines for young children. A number of animal FI-RSV studies suggest various immune mechanisms behind ERD. However, other than limited data from the original FI-RSV trial, there is no information on the human ERD-associated responses. An in vitro model with human blood specimens may shed light on the immune memory responses likely responsible for ERD. Memory T cell responses to an antigen are guided by the innate responses, particularly dendritic cells that present an antigen in conjunction with co-stimulatory molecules and cytokine signaling. Our in vitro model involves human monocyte derived dendritic cells (moDC) and allogenic T cell cultures to assess innate responses that direct T cell responses. Using this model, we evaluated human responses to live RSV, FI-RSV, and subunit RSV G vaccines (G-containing virus-like particles, G-VLP). Similar to findings in animal studies, FI-RSV induced prominent Th2/Th17-biased responses with deficient type-1 responses compared to live virus. Responses to G-VLPs were similar to live virus, i.e. biased towards a Th1 and not a Th2/Th17. Also mutating CX3C motif in G gave a more pronounced moDC responses associated with type-1 T cell responses. This in vitro model identifies human immune responses likely associated with ERD and provides another pre-clinical tool to assess the safety of RSV vaccines.


Assuntos
Vacinas contra Vírus Sincicial Respiratório/efeitos adversos , Vacinas contra Vírus Sincicial Respiratório/imunologia , Animais , Apresentação do Antígeno , Antígenos Virais/imunologia , Pré-Escolar , Células Dendríticas/imunologia , Células Dendríticas/virologia , Humanos , Imunidade Inata , Memória Imunológica , Técnicas In Vitro , Lactente , Modelos Imunológicos , Infecções por Vírus Respiratório Sincicial/etiologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sincicial Respiratório Humano/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Vacinas de Subunidades/efeitos adversos , Vacinas de Subunidades/imunologia
4.
PLoS Pathog ; 15(10): e1007984, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31622448

RESUMO

Human respiratory syncytial virus (RSV) is the most important cause of acute lower respiratory tract disease in infants worldwide. As a first line of defense against respiratory infections, innate immune responses, including the production of type I and III interferons (IFNs), play an important role. Upon infection with RSV, multiple pattern recognition receptors (PRRs) can recognize RSV-derived pathogen-associated molecular patterns (PAMPs) and mount innate immune responses. Retinoic-acid-inducible gene-I (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) have been identified as important innate receptors to mount type I IFNs during RSV infection. However, type I IFN levels remain surprisingly low during RSV infection despite strong viral replication. The poor induction of type I IFNs can be attributed to the cooperative activity of 2 unique, nonstructural (NS) proteins of RSV, i.e., NS1 and NS2. These viral proteins have been shown to suppress both the production and signaling of type I and III IFNs by counteracting a plethora of key host innate signaling proteins. Moreover, increasing numbers of IFN-stimulated genes (ISGs) are being identified as targets of the NS proteins in recent years, highlighting an underexplored protein family in the identification of NS target proteins. To understand the diverse effector functions of NS1 and NS2, Goswami and colleagues proposed the hypothesis of the NS degradasome (NSD) complex, a multiprotein complex made up of, at least, NS1 and NS2. Furthermore, the crystal structure of NS1 was resolved recently and, remarkably, identified NS1 as a structural paralogue of the RSV matrix protein. Unfortunately, no structural data on NS2 have been published so far. In this review, we briefly describe the PRRs that mount innate immune responses upon RSV infection and provide an overview of the various effector functions of NS1 and NS2. Furthermore, we discuss the ubiquitination effector functions of NS1 and NS2, which are in line with the hypothesis that the NSD shares features with the canonical 26S proteasome.


Assuntos
Imunidade Inata , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas não Estruturais Virais/metabolismo , Humanos , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Transdução de Sinais , Ubiquitinação , Replicação Viral
5.
Nat Commun ; 10(1): 4595, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31597913

RESUMO

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.


Assuntos
Adenosina/análogos & derivados , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Replicação Viral/imunologia , Células A549 , Adenosina/genética , Adenosina/imunologia , Adenosina/metabolismo , Animais , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Feminino , Células HeLa , Humanos , Masculino , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/patogenicidade , Sigmodontinae , Regulação para Cima/imunologia , Vacinas Atenuadas/imunologia , Células Vero , Virulência/genética , Virulência/imunologia , Replicação Viral/genética
6.
Nat Commun ; 10(1): 4153, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515478

RESUMO

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Sequência Conservada , Glicoproteínas/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Linfócitos B/imunologia , Sítios de Ligação , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Humanos , Memória Imunológica , Modelos Moleculares , Ligação Proteica , Sigmodontinae
7.
Internist (Berl) ; 60(11): 1146-1150, 2019 Nov.
Artigo em Alemão | MEDLINE | ID: mdl-31486858

RESUMO

Respiratory syncytial virus (RSV) is worldwide a very important virus leading to infection of the respiratory system. In particular preterm babies, infants and elderly adults are prone to developing severe diseases such as bronchiolitis or pneumonia, which require intensive care and cause increased mortality. Although RSV is rapidly detected, preventive and therapeutic measures are limited. New antivirals are already in clinical trials.


Assuntos
Antivirais/administração & dosagem , Bronquiolite/diagnóstico , Bronquiolite/prevenção & controle , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sincicial Respiratório Humano/isolamento & purificação , Adulto , Idoso , Antivirais/uso terapêutico , Bronquiolite/tratamento farmacológico , Bronquiolite/virologia , Bronquiolite Viral/diagnóstico , Bronquiolite Viral/virologia , Humanos , Lactente , Recém-Nascido , Pneumonia/diagnóstico , Pneumonia/virologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/imunologia , Fatores de Risco
8.
Science ; 365(6452): 505-509, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31371616

RESUMO

Technologies that define the atomic-level structure of neutralization-sensitive epitopes on viral surface proteins are transforming vaccinology and guiding new vaccine development approaches. Previously, iterative rounds of protein engineering were performed to preserve the prefusion conformation of the respiratory syncytial virus (RSV) fusion (F) glycoprotein, resulting in a stabilized subunit vaccine candidate (DS-Cav1), which showed promising results in mice and macaques. Here, phase I human immunogenicity data reveal a more than 10-fold boost in neutralizing activity in serum from antibodies targeting prefusion-specific surfaces of RSV F. These findings represent a clinical proof of concept for structure-based vaccine design, suggest that development of a successful RSV vaccine will be feasible, and portend an era of precision vaccinology.


Assuntos
Imunogenicidade da Vacina , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/química , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Mapeamento de Epitopos , Humanos , Pessoa de Meia-Idade , Adulto Jovem
9.
Jpn J Infect Dis ; 72(6): 394-398, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31257240

RESUMO

We evaluated two currently available rapid antigen detection tests (RADTs) for Respiratory syncytial virus (RSV), Sofia® RSV FIA and BinaxNOW RSV Card (BinaxNOW). Between November 2017 and February 2018, 395 nasopharyngeal swabs were collected from children diagnosed with acute respiratory infections. The swabs were evaluated using the aforementioned RADTs, the reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR), and the direct immunofluorescence assay (DFA). The sensitivity of Sofia® RSV FIA (80.82%) was significantly higher than that of BinaxNOW (53.42%) when RT-qPCR was used as the standard. This was confirmed with DFA. The sensitivities of Sofia® RSV FIA (85.4% [41/48]) and BinaxNOW (58.3% [28/48]) were higher for RSV A than for RSV B (69.6% [16/23] and 43.5% [10/23], respectively). The optimal critical cycle threshold (Ct) values on RT-qPCR that correlated with Sofia® RSV FIA and BinaxNOW were 24 and 22, respectively. The kappa value for Sofia® RSV FIA and RT-qPCR was 0.962 in patients who were two years old or younger, but 0.648 in those who were more than two years old. Thus, Sofia® RSV FIA is more sensitive than BinaxNOW; its results were affected by the RSV viral strain and load. Sofia® RSV FIA is more effective in children who are ≤ 2 years old than in those who are > 2 years old.


Assuntos
Antígenos Virais/isolamento & purificação , Técnica Direta de Fluorescência para Anticorpo/normas , Kit de Reagentes para Diagnóstico/normas , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/isolamento & purificação , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Nasofaringe/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/imunologia , Infecções Respiratórias/diagnóstico , Sensibilidade e Especificidade
10.
PLoS Pathog ; 15(7): e1007944, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31306469

RESUMO

The respiratory syncytial virus (RSV) fusion (F) glycoprotein is a major target of neutralizing antibodies arising from natural infection, and antibodies that specifically bind to the prefusion conformation of RSV F generally demonstrate the greatest neutralization potency. Prefusion-stabilized RSV F variants have been engineered as vaccine antigens, but crystal structures of these variants have revealed conformational differences in a key antigenic site located at the apex of the trimer, referred to as antigenic site Ø. Currently, it is unclear if flexibility in this region is an inherent property of prefusion RSV F or if it is related to inadequate stabilization of site Ø in the engineered variants. Therefore, we set out to investigate the conformational flexibility of antigenic site Ø, as well as the ability of the human immune system to recognize alternative conformations of this site, by determining crystal structures of prefusion RSV F bound to neutralizing human-derived antibodies AM22 and RSD5. Both antibodies bound with high affinity and were specific for the prefusion conformation of RSV F. Crystal structures of the complexes revealed that the antibodies recognized distinct conformations of antigenic site Ø, each diverging at a conserved proline residue located in the middle of an α-helix. These data suggest that antigenic site Ø exists as an ensemble of conformations, with individual antibodies recognizing discrete states. Collectively, these results have implications for the refolding of pneumovirus and paramyxovirus fusion proteins and should inform development of prefusion-stabilized RSV F vaccine candidates.


Assuntos
Antígenos Virais/química , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Sítios de Ligação/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Prolina/química , Conformação Proteica , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais de Fusão/genética
11.
Virology ; 534: 1-13, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31163351

RESUMO

Alum adjuvanted formalin-inactivated respiratory syncytial virus (RSV) vaccination resulted in enhanced respiratory disease in young children upon natural infection. Here, we investigated the adjuvant effects of monophosphoryl lipid A (MPL) and oligodeoxynucleotide CpG (CpG) on vaccine-enhanced respiratory disease after fusion (F) protein prime vaccination and RSV challenge in infant and adult mouse models. Combination CpG + MPL adjuvant in RSV F protein single dose priming of infant and adult age mice was found to promote the induction of IgG2a isotype antibodies and neutralizing activity, and lung viral clearance after challenge. CpG + MPL adjuvanted F protein (Fp) priming of infant and adult age mice was effective in avoiding lung histopathology, in reducing interleukin-4+ CD4 T cells and cellular infiltration of monocytes and neutrophils after RSV challenge. This study suggests that combination CpG and MPL adjuvant in RSV subunit vaccination might contribute to priming protective immune responses and preventing inflammatory RSV disease after infection.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Pulmão/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/administração & dosagem , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Humanos , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/fisiologia , Vacinação , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia
12.
Biomolecules ; 9(5)2019 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035368

RESUMO

Human respiratory syncytial virus (HRSV) infection is a common cause of severe lower respiratory tract diseases such as bronchiolitis and pneumonia. Both virus replication and the associated inflammatory immune response are believed to be behind these pathologies. So far, no vaccine or effective treatment is available for this viral infection. With the aim of finding new strategies to counteract HRSV replication and modulate the immune response, specific small interfering RNAs (siRNAs) were generated targeting the mRNA coding for the viral fusion (F) protein or nucleoprotein (N), or for two proteins involved in intracellular immune signaling, which are named tripartite motif-containing protein 25 (TRIM25) and retinoic acid-inducible gene-I (RIG-I). Furthermore, two additional bispecific siRNAs were designed that silenced F and TRIM25 (TRIM25/HRSV-F) or N and RIG-I (RIG-I/HRSV-N) simultaneously. All siRNAs targeting N or F, but not those silencing TRIM25 or RIG-I alone, significantly reduced viral titers. However, while siRNAs targeting F inhibited only the expression of the F mRNA and protein, the siRNAs targeting N led to a general inhibition of viral mRNA and protein expression. The N-targeting siRNAs also induced a drastic decrease in the expression of genes of the innate immune response. These results show that both virus replication and the early innate immune response can be regulated by targeting distinct viral products with siRNAs, which may be related to the different role of each protein in the life cycle of the virus.


Assuntos
Imunidade Inata , RNA Interferente Pequeno/metabolismo , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/fisiologia , Replicação Viral/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica , Inativação Gênica , Genoma Viral , Humanos , Imunidade Inata/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Proteínas Virais/genética , Proteínas Virais/metabolismo
13.
Sci Immunol ; 4(35)2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101673

RESUMO

Effective vaccines inducing lifelong protection against many important infections such as respiratory syncytial virus (RSV), HIV, influenza virus, and Epstein-Barr virus (EBV) are not yet available despite decades of research. As an alternative to a protective vaccine, we developed a genetic engineering strategy in which CRISPR-Cas9 was used to replace endogenously encoded antibodies with antibodies targeting RSV, HIV, influenza virus, or EBV in primary human B cells. The engineered antibodies were expressed efficiently in primary B cells under the control of endogenous regulatory elements, which maintained normal antibody expression and secretion. Using engineered mouse B cells, we demonstrated that a single transfer of B cells engineered to express an antibody against RSV resulted in potent and durable protection against RSV infection in RAG1-deficient mice. This approach offers the opportunity to achieve sterilizing immunity against pathogens for which traditional vaccination has failed to induce or maintain protective antibody responses.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Engenharia Metabólica/métodos , Infecções por Vírus Respiratório Sincicial/terapia , Vírus Sincicial Respiratório Humano/imunologia , Células 3T3 , Transferência Adotiva/métodos , Animais , Sistemas CRISPR-Cas , Feminino , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Infecções por Vírus Respiratório Sincicial/virologia
14.
Nat Commun ; 10(1): 2218, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101811

RESUMO

RSV infection is typically associated with secondary bacterial infection. We hypothesise that the local airway immune response to RSV has incidental antibacterial effects. Using coordinated proteomics and metagenomics analysis we simultaneously analysed the microbiota and proteomes of the upper airway and determined direct antibacterial activity in airway secretions of RSV-infected children. Here, we report that the airway abundance of Streptococcus was higher in samples collected at the time of RSV infection compared with samples collected one month later. RSV infection is associated with neutrophil influx into the airway and degranulation and is marked by overexpression of proteins with known antibacterial activity including BPI, EPX, MPO and AZU1. Airway secretions of children infected with RSV, have significantly greater antibacterial activity compared to RSV-negative controls. This RSV-associated, neutrophil-mediated antibacterial response in the airway appears to act as a regulatory mechanism that modulates bacterial growth in the airways of RSV-infected children.


Assuntos
Infecções Bacterianas/imunologia , Neutrófilos/imunologia , Mucosa Respiratória/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Infecções Bacterianas/microbiologia , Degranulação Celular/imunologia , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Quênia , Metagenômica/métodos , Microbiota/imunologia , Proteômica/métodos , Mucosa Respiratória/citologia , Mucosa Respiratória/microbiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Streptococcus/imunologia , Streptococcus/isolamento & purificação
15.
Nat Commun ; 10(1): 2331, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133680

RESUMO

Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid ß-peptide (Aß42), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Herpesvirus Humano 1/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Fragmentos de Peptídeos/metabolismo , Coroa de Proteína/imunologia , Vírus Sincicial Respiratório Humano/patogenicidade , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Doença de Alzheimer/virologia , Animais , Líquido da Lavagem Broncoalveolar/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Voluntários Saudáveis , Herpes Simples/sangue , Herpes Simples/imunologia , Herpes Simples/patologia , Herpesvirus Humano 1/imunologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Agregados Proteicos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/imunologia , Células Vero
16.
Virus Res ; 266: 58-68, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31004621

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization worldwide each year and there is presently no licensed vaccine to prevent severe RSV infections. Two major RSV glycoproteins, attachment (G) and fusion (F) protein, regulate viral replication and both proteins contain potential glycosylation sites which are highly variable for the G protein and conserved for the F protein among virus isolates. The RSV F sequence possesses five N-glycosylation sites located in the F2 subunit (N27 and N70), the p27 peptide (N116 and N126) and the F1 subunit (N500). The importance of RSV F N-glycosylation in virus replication and immunogenicity is not yet fully understood, and a better understanding may provide new insights for vaccine development. By using a BAC-based reverse genetics system, recombinant viruses expressing F proteins with loss of N-glycosylation sites were made. Mutant viruses with single N-glycosylation sites removed could be recovered, while this was not possible with the mutant with all N-glycosylation sites removed. Although the individual RSV F N-glycosylation sites were shown not to be essential for viral replication, they do contribute to the efficiency of in vitro and in vivo viral infection. To evaluate the role of N-glycosylation sites on RSV F antigenicity, serum antibody titers were determined after infection of BALB/c mice with RSV expressing the glycomutant F proteins. Infection with recombinant virus lacking the N-glycosylation site at position N116 (RSV F N116Q) resulted in significant higher neutralizing antibody titers compared to RSV F WT infection, which is surprising since this N-glycan is present in the p27 peptide which is assumed to be absent from the mature F protein in virions. Thus, single or combined RSV F glycomutations which affect virus replication and fusogenicity, and which may induce enhanced antibody responses upon immunization could have the potential to improve the efficacy of RSV LAV approaches.


Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Vírus Sincicial Respiratório Humano/patogenicidade , Proteínas Virais de Fusão/metabolismo , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Células Gigantes/virologia , Glicosilação , Humanos , Imunização , Imunogenicidade da Vacina , Camundongos Endogâmicos BALB C , Mutação , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/patologia , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/imunologia , Células Vero , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Replicação Viral
17.
Infect Genet Evol ; 71: 166-178, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30946992

RESUMO

HRSV is a principle cause of infant hospitalization, childhood wheezing and a common pathogen in the elderly. Limited information exists regarding HRSV genotypes in Iran. In order to better understand HRSV strain diversity, we performed an in-depth evaluation of the genetic variability of the HRSV F protein detected in children under two years of age that, presented with acute respiratory symptoms during 2015-2016 in Tehran. A total of 180 nasopharyngeal swabs were evaluated. The HRSV positive samples were genotyped for G and F gene sequences using RT-PCR and sequencing methods. Phylogenetic analysis was performed using the neighbor-joining and maximum likelihood methods. Genetic and antigenic characteristics of the F gene, nucleotide and amino acids in significant positions and immune system binding regions, as well as the p-distance, positive/negative selection site, linear epitopes and glycosylation sites were investigated in all selected sequences. Among the 83 HRSV positive samples, the Fifty-five cases were successfully sequenced. All of them were classified as subgroup A and belonged to the ON-1 genotype, which possessed 72-nt duplication in the G gene. This study is the first report on the emergence of ON-1 in Iran. ON-1 Iranian sequences clustered in three lineages according to virus fusion (F) gene variations. F gene sequence analysis showed that all genetic changes in the isolates from Iran were base substitutions and no deletion/insertions were identified. The low dN/dS ratio and lack of positively selected sites showed that the fusion genes found in the strains from Iran are not under host selective pressure. Continuing and long-term molecular epidemiological surveys for early detection of circulating and newly emerging genotypes are necessary to gain a better understanding of their epidemic potential.


Assuntos
Vírus Sincicial Respiratório Humano/genética , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética , Variação Antigênica , Feminino , Genótipo , Humanos , Lactente , Irã (Geográfico)/epidemiologia , Masculino , Epidemiologia Molecular , Filogenia , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas Virais de Fusão/imunologia
18.
Enferm. infecc. microbiol. clín. (Ed. impr.) ; 37(4): 251-255, abr. 2019. tab
Artigo em Inglês | IBECS | ID: ibc-189205

RESUMO

INTRODUCTION: Passive transplacental immunity against respiratory syncytial virus (RSV) appears to mediate in the protection of the infant for the first 6 months of life. Lower environmental exposure in pregnant women to RSV epidemic may influence the susceptibility of these infants to infection by lowering the levels of antibodies that are transferred to the fetus. OBJECTIVES: To contrast the risk of severe disease progression in infants with acute bronchiolitis by RSV, according to the mother's level of exposure to epidemic. METHOD: Retrospective cohort study of previously healthy infants with RSV-acute bronchiolitis during 5 epidemics was made. We compared the severity of the infection in those born during the period of risk (when is less likely the mother's exposure to epidemic and the transfer of antibodies to the fetus: October 15th-December 15 th in our latitude) with the rest of acute bronchiolitis. Bivariate analysis was performed regarding birth in period of risk and the rest of variables, using the Chi-square test. Multivariate logistic regression analysis was performed to study possible classical confounding factors. RESULTS: 695 infants were included in the study. 356 infants were born during the period of risk. Of the 56 patients requiring admission to PICU, 40 of them (71.4%) were born in this period (p = 0.002). In the multivariate analysis, the birth in the period of risk showed a 6.5 OR (95% CI: 2.13-19.7) independently of the rest of variables. CONCLUSIONS: The worst clinical disease progression of the acute bronchiolitis by the RSV in less than 6 months age is related to lower exposure of the pregnant woman to the RSV epidemic


INTRODUCCIÓN: La inmunidad pasiva transplacentaria contra el virus respiratorio sincitial (VRS) parece mediar en la protección del lactante durante los primeros 6 meses de vida. La menor exposición ambiental en embarazadas a la epidemia del VRS puede influir en la susceptibilidad de estos niños a la infección al disminuir los niveles de anticuerpos que se transfieren al feto. OBJETIVOS: Contrastar el riesgo de progresión grave de la enfermedad en los lactantes con bronquiolitis aguda por VRS, de acuerdo con el nivel de exposición de la madre a la epidemia. MÉTODO: Se realizó un estudio de cohortes retrospectivo de recién nacidos previamente sanos con bronquiolitis aguda por VRS durante 5 epidemias. Comparamos la gravedad de la infección en los nacidos durante el período de riesgo (cuando es menos probable la exposición de la madre a la epidemia en nuestra latitud y la transferencia de anticuerpos al feto: del 15 de octubre al 15 de diciembre) con el resto de las bronquiolitis agudas. El análisis bivariante se realizó con respecto al nacimiento en el período de riesgo y el resto de las variables, utilizando la prueba de Chi-cuadrado. Posteriormente un análisis de regresión logística multivariable para estudiar los posibles factores de confusión. RESULTADOS: Seiscientos noventa y cinco bebés fueron incluidos en el estudio. Trescientos cincuenta y seis bebés nacieron durante el período de riesgo. De los 56 pacientes que requieren ingreso en la UCIP, 40 de ellos (71,4%) nacieron en este período (p = 0,002). En el análisis multivariante, el nacimiento en el período de riesgo mostró una OR de 6,5 (IC 95%: 2,13-19,7) independientemente del resto de las variables. CONCLUSIONES: La evolución más grave de la enfermedad clínica de la bronquiolitis aguda por el VRS en menores de 6 meses de edad se relaciona con una menor exposición de la embarazada a la epidemia del VRS


Assuntos
Humanos , Masculino , Feminino , Lactente , Bronquiolite Viral/imunologia , Bronquiolite Viral/virologia , Imunidade Materno-Adquirida/imunologia , Gravidez/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Doença Aguda , Infecções por Vírus Respiratório Sincicial/epidemiologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Estudos de Coortes
19.
Hum Vaccin Immunother ; 15(10): 2466-2474, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30852939

RESUMO

Background: Infections with respiratory syncytial virus (RSV) cause significant morbidity and hospitalization in older adults. We studied the humoral, mucosal and B cell responses of an investigational adjuvanted RSV sF vaccine, MEDI7510, in older adults. Methods: In a substudy of a randomized (1:1), double-blind, placebo-controlled study of MEDI7510 in adults ≥60 years of age, we collected blood and nasal secretions at days 0, 8, 29, 91 and 180 post-vaccination to measure F-specific IgG and IgA antibodies by ELISA, and plasmablasts and memory B cells by IgA/IgG dual-color fluorospot. Results: The 27 vaccine- and 18 placebo-recipients had a mean age of 73 years and included 24 women. Among vaccinees, 93% had significant increases in F-specific plasma IgG 85% had increased plasma IgA; 74% had increased nasal IgG and 26% nasal IgA; 93% had IgG and 89% IgA plasmablasts on Day 8 post-immunization; and 82% had IgG and 7.4% IgA memory B cell responses to the vaccine. Vaccinees <70 years of age and women had the highest responses to the vaccine. Conclusions: This adjuvanted vaccine generated robust humoral immune responses in older adults, including RSV F-specific systemic and mucosal antibodies and memory B cells. Nevertheless, age ≥70 years was associated with decreased immunogenicity of the adjuvanted vaccine.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Método Duplo-Cego , Feminino , Humanos , Imunidade Humoral , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem
20.
Nat Commun ; 10(1): 1126, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850611

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of hospitalization in infants and young children. Although it is widely agreed that an RSV vaccine should induce both mucosal and systemic antibody responses, little is known about the B cell response to RSV in mucosa-associated lymphoid tissues. Here, we analyze this response by isolating 806 RSV F-specific antibodies from paired adenoid and peripheral blood samples from 4 young children. Overall, the adenoid-derived antibodies show higher binding affinities and neutralization potencies compared to antibodies isolated from peripheral blood. Approximately 25% of the neutralizing antibodies isolated from adenoids originate from a unique population of IgM+ and/or IgD+ memory B cells that contain a high load of somatic mutations but lack expression of classical memory B cell markers. Altogether, the results provide insight into the local B cell response to RSV and have implications for the development of vaccines that stimulate potent mucosal responses.


Assuntos
Tonsila Faríngea/imunologia , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Linfócitos B/imunologia , Imunidade nas Mucosas , Leucócitos Mononucleares/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Tonsila Faríngea/virologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Linfócitos B/virologia , Biomarcadores/metabolismo , Pré-Escolar , Clonagem Molecular , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Imunoglobulina D/biossíntese , Imunoglobulina M/biossíntese , Leucócitos Mononucleares/virologia , Masculino , Mutação , Especificidade de Órgãos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
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