RESUMO
Acute respiratory infection by influenza virus is a persistent and pervasive public health problem. Antiviral innate immunity initiated by type I interferon (IFN) is the first responder to pathogen invasion and provides the first line of defense. We discovered that Axin1, a scaffold protein, was reduced during influenza virus infection. We also found that overexpression of Axin1 and the chemical stabilizer of Axin1, XAV939, reduced influenza virus replication in lung epithelial cells. This effect was also observed with respiratory syncytial virus and vesicular stomatitis virus. Axin1 boosted type I IFN response to influenza virus infection and activated JNK/c-Jun and Smad3 signaling. XAV939 protected mice from influenza virus infection. Thus, our studies provide new mechanistic insights into the regulation of the type I IFN response and present a new potential therapeutic of targeting Axin1 against influenza virus infection.
Assuntos
Proteína Axina , Influenza Humana , Interferons , Animais , Humanos , Camundongos , Proteína Axina/metabolismo , Células Epiteliais , Imunidade Inata , Influenza Humana/imunologia , Influenza Humana/metabolismo , Interferons/metabolismo , Replicação ViralRESUMO
Respiratory syncytial virus (RSV) has been reported to use CX3CR1 in vitro as a receptor on cultured primary human airway epithelial cultures. To evaluate CX3CR1 as the receptor for RSV in vivo, we used the cotton rat animal model because of its high permissiveness for RSV infection. Sequencing the cotton rat CX3CR1 gene revealed 91% amino acid similarity to human CX3CR1. Previous work found that RSV binds to CX3CR1 via its attachment glycoprotein (G protein) to infect primary human airway cultures. To determine whether CX3CR1-G protein interaction is necessary for RSV infection, recombinant RSVs containing mutations in the CX3CR1 binding site of the G protein were tested in cotton rats. In contrast to wild-type virus, viral mutants did not grow in the lungs of cotton rats. When RSV was incubated with an antibody blocking the CX3CR1 binding site of G protein and subsequently inoculated intranasally into cotton rats, no virus was found in the lungs 4 days postinfection. In contrast, growth of RSV was not affected after preincubation with heparan sulfate (the receptor for RSV on immortalized cell lines). A reduction in CX3CR1 expression in the cotton rat lung through the use of peptide-conjugated morpholino oligomers led to a 10-fold reduction in RSV titers at day 4 postinfection. In summary, these results indicate that CX3CR1 functions as a receptor for RSV in cotton rats and, in combination with data from human airway epithelial cell cultures, strongly suggest that CX3CR1 is a primary receptor for naturally acquired RSV infection. IMPORTANCE The knowledge about a virus receptor is useful to better understand the uptake of a virus into a cell and potentially develop antivirals directed against either the receptor molecule on the cell or the receptor-binding protein of the virus. Among a number of potential receptor proteins, human CX3CR1 has been demonstrated to act as a receptor for respiratory syncytial virus (RSV) on human epithelial cells in tissue culture. Here, we report that the cotton rat CX3CR1, which is similar to the human molecule, acts as a receptor in vivo. This study strengthens the argument that CX3CR1 is a receptor molecule for RSV.
Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Receptores Virais/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Animais , Anticorpos Antivirais/farmacologia , Sítios de Ligação , Receptor 1 de Quimiocina CX3C/antagonistas & inibidores , Receptor 1 de Quimiocina CX3C/química , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/virologia , Heparitina Sulfato/metabolismo , Humanos , Mutação , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/metabolismo , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , Sigmodontinae , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral/genéticaRESUMO
Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children, but no vaccine is currently available. Live-attenuated vaccines represent an attractive immunization approach; however, balancing attenuation while retaining sufficient immunogenicity and efficacy has prevented the successful development of such a vaccine. Recently, a recombinant RSV strain lacking the gene that encodes the matrix (M) protein (RSV M-null) was developed. The M protein is required for virion assembly following infection of a host cell but is not necessary for either genome replication or gene expression. Therefore, infection with RSV M-null produces all viral proteins except M but does not generate infectious virus progeny, resulting in a single-cycle infection. We evaluated RSV M-null as a potential vaccine candidate by determining its pathogenicity, immunogenicity, and protective capacity in BALB/c mice compared with its recombinant wild-type control virus (RSV recWT). RSV M-null-infected mice exhibited significantly reduced lung viral titers, weight loss, and pulmonary dysfunction compared with mice infected with RSV recWT. Despite its attenuation, RSV M-null infection induced robust immune responses of similar magnitude to that elicited by RSV recWT. Additionally, RSV M-null infection generated serum Ab and memory T cell responses that were similar to those induced by RSV recWT. Importantly, RSV M-null immunization provided protection against secondary viral challenge by reducing lung viral titers as efficiently as immunization with RSV recWT. Overall, our results indicate that RSV M-null combines attenuation with high immunogenicity and efficacy and represents a promising novel live-attenuated RSV vaccine candidate.
Assuntos
Pulmão/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/fisiologia , Linfócitos T/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/metabolismo , Resistência à Doença , Imunidade Celular , Memória Imunológica , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinação , Proteínas Virais/genéticaRESUMO
Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology. IMPORTANCE: hRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines.
Assuntos
Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas Virais/imunologia , Antígenos Virais/química , Antígenos Virais/genética , Linhagem Celular , Humanos , Microscopia Eletrônica de Varredura , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas contra Vírus Sincicial Respiratório/química , Vacinas contra Vírus Sincicial Respiratório/genética , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/fisiologia , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/ultraestrutura , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Proteínas Virais/química , Proteínas Virais/genética , Montagem de VírusRESUMO
Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a "non-neutralizing" monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization.
Assuntos
Interações Hospedeiro-Parasita/fisiologia , Receptores de Quimiocinas/metabolismo , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Animais , Receptor 1 de Quimiocina CX3C , Linhagem Celular , Células Cultivadas , Proteínas de Ligação ao GTP/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Transfecção , Proteínas Virais/metabolismo , Internalização do VírusRESUMO
Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and â¼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.
Assuntos
Células Epiteliais/metabolismo , Receptores de Quimiocinas/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Motivos de Aminoácidos , Receptor 1 de Quimiocina CX3C , Linhagem Celular , Células Epiteliais/virologia , Humanos , Ligação Proteica , Receptores de Quimiocinas/química , Receptores de Quimiocinas/genética , Infecções por Vírus Respiratório Sincicial/genética , Vírus Sincicial Respiratório Humano/genética , Sistema Respiratório/citologia , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
The human respiratory syncytial virus (HRSV) fusion (F) protein cytoplasmic tail (CT) and matrix (M) protein are key mediators of viral assembly, but the underlying mechanisms are poorly understood. A complementation assay was developed to systematically examine the role of the F protein CT in infectious virus production. The ability of F mutants with alanine substitutions in the CT to complement an F-null virus in generating infectious progeny was quantitated by flow cytometry. Two CT regions with impact on infectious progeny production were identified: residues 557 to 566 (CT-R1) and 569 to 572 (CT-R2). Substitutions in CT-R1 decreased infectivity by 40 to 85% and increased the level of F-induced cell-cell fusion but had little impact on assembly of viral surface filaments, which are believed to be virions. Substitutions in CT-R2, as well as deletion of the entire CT, abrogated infectious progeny production and impaired viral filament formation. However, CT-R2 mutations did not block but rather delayed the formation of viral filaments, which continued to form at a low rate and contained the viral M protein and nucleoprotein (N). Microscopy analysis revealed that substitutions in CT-R2 but not CT-R1 led to accumulation of M and F proteins within and at the perimeter of viral inclusion bodies (IBs), respectively. The accumulation of M and F at IBs and coincident strong decrease in filament formation and infectivity upon CT-R2 mutations suggest that F interaction with IBs is an important step in the virion assembly process and that CT residues 569 to 572 act to facilitate release of M-ribonucleoprotein complexes from IBs.
Assuntos
Células Epiteliais/virologia , Corpos de Inclusão Viral/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/patogenicidade , Proteínas Virais de Fusão/metabolismo , Animais , Fusão Celular , Células Cultivadas , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Fusão de Membrana , Mutação/genética , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/metabolismo , Células Vero , Proteínas Virais de Fusão/genética , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab')2 form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.
Assuntos
Células Epiteliais/imunologia , Leucócitos Mononucleares/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais/imunologia , Imunidade Adaptativa , Motivos de Aminoácidos , Receptor 1 de Quimiocina CX3C , Quimiocinas CX3C/imunologia , Células Epiteliais/virologia , Humanos , Imunidade Inata , Interferons/genética , Interferons/imunologia , Leucócitos Mononucleares/virologia , Receptores de Quimiocinas/imunologia , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Respiratory syncytial virus (RSV) is a major cause of severe respiratory tract disease worldwide, and a pediatric vaccine is not available. We generated a filamentous RSV-based virus-like particle (VLP) that presents the central conserved region of the attachment protein G. This was achieved by co-expressing the matrix protein, phosphoprotein, nucleoprotein, and a hybrid fusion protein in which the F ectodomain was replaced with the G central region (GCR). The latter is relatively conserved and contains a receptor binding site and hence is a logical vaccine target. The immunogenicity and efficacy of the resulting VLP, termed VLP-GCR, were examined in mice using intranasal application without adjuvant. VLP-GCR induced substantial anti-N antibody levels but very low anti-G antibody levels, even after three vaccinations. In contrast, a VLP presenting prefusion-stabilized fusion (preF) protein instead of GCR induced both high anti-F and anti-nucleoprotein antibody levels, suggesting that our GCR antigen was poorly immunogenic. Challenge of VLP-GCR-vaccinated mice caused increased weight loss and lung pathology, and both VLPs induced mucus in the lungs. Thus, neither VLP is suitable as a vaccine for RSV-naive individuals. However, VLP-preF enhanced the proportion of preF antibodies and could serve as a multi-antigen mucosal booster vaccine in the RSV-experienced population.
Assuntos
Anticorpos Antivirais , Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vacinas de Partículas Semelhantes a Vírus , Animais , Feminino , Humanos , Camundongos , Administração Intranasal , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Pulmão/virologia , Pulmão/patologia , Pulmão/imunologia , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/genética , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/genética , Vacinação , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/administração & dosagem , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/genética , Redução de PesoRESUMO
An experimental system was developed to generate infectious human respiratory syncytial virus (HRSV) lacking matrix (M) protein expression (M-null virus) from cDNA. The role of the M protein in virus assembly was then examined by infecting HEp-2 and Vero cells with the M-null virus and assessing the impact on infectious virus production and viral protein trafficking. In the absence of M, the production of infectious progeny was strongly impaired. Immunofluorescence (IF) microscopy analysis using antibodies against the nucleoprotein (N), attachment protein (G), and fusion protein (F) failed to detect the characteristic virus-induced cell surface filaments, which are believed to represent infectious virions. In addition, a large proportion of the N protein was detected in viral replication factories termed inclusion bodies (IBs). High-resolution analysis of the surface of M-null virus-infected cells by field emission scanning electron microscopy (SEM) revealed the presence of large areas with densely packed, uniformly short filaments. Although unusually short, these filaments were otherwise similar to those induced by an M-containing control virus, including the presence of the viral G and F proteins. The abundance of the short, stunted filaments in the absence of M indicates that M is not required for the initial stages of filament formation but plays an important role in the maturation or elongation of these structures. In addition, the absence of mature viral filaments and the simultaneous increase in the level of the N protein within IBs suggest that the M protein is involved in the transport of viral ribonucleoprotein (RNP) complexes from cytoplasmic IBs to sites of budding.
Assuntos
Vírus Sincicial Respiratório Humano/metabolismo , Proteínas da Matriz Viral/metabolismo , Animais , Linhagem Celular , Extensões da Superfície Celular/ultraestrutura , Chlorocebus aethiops , Códon , Expressão Gênica , Ordem dos Genes , Humanos , Mutação , Fases de Leitura Aberta/genética , Transporte Proteico , Vírus Sincicial Respiratório Humano/genética , Proteínas da Matriz Viral/genética , Replicação Viral/genéticaRESUMO
Introduction: Respiratory infections remain a leading global health concern. Models that recapitulate the cellular complexity of the lower airway of humans will provide important information about how the immune response reflects the interactions between diverse cell types during infection. We developed a 3D human tissue-engineered lung model (3D-HTLM) composed of primary human pulmonary epithelial and endothelial cells with added blood myeloid cells that allows assessment of the innate immune response to respiratory infection. Methods: The 3D-HTLM consists of small airway epithelial cells grown at air-liquid interface layered on fibroblasts within a collagen matrix atop a permeable membrane with pulmonary microvascular endothelial cells layered underneath. After the epithelial and endothelial layers had reached confluency, an enriched blood monocyte population, containing mostly CD14+ monocytes (Mo) with minor subsets of CD1c+ classical dendritic cells (cDC2s), monocyte-derived dendritic cells (Mo-DCs), and CD16+ non-classical monocytes, was added to the endothelial side of the model. Results: Immunofluorescence imaging showed the myeloid cells migrate through and reside within each layer of the model. The myeloid cell subsets adapted to the lung environment in the 3D-HTLM, with increased proportions of the recovered cells expressing lung tissue resident markers CD206, CD169, and CD163 compared with blood myeloid cells, including a population with features of alveolar macrophages. Myeloid subsets recovered from the 3D-HTLM displayed increased expression of HLA-DR and the co-stimulatory markers CD86, CD40, and PDL1. Upon stimulation of the 3D-HTLM with the toll-like receptor 4 (TLR4) agonist bacterial lipopolysaccharide (LPS), the CD31+ endothelial cells increased expression of ICAM-1 and the production of IL-10 and TNFα was dependent on the presence of myeloid cells. Challenge with respiratory syncytial virus (RSV) led to increased expression of macrophage activation and antiviral pathway genes by cells in the 3D-HTLM. Discussion: The 3D-HTLM provides a lower airway environment that promotes differentiation of blood myeloid cells into lung tissue resident cells and enables the study of respiratory infection in a physiological cellular context.
RESUMO
The human respiratory syncytial virus (RSV) is a major cause of severe respiratory tract disease, and a vaccine is not available. We previously reported a novel live vaccine expressing prefusion-stabilized fusion protein (preF) in place of the native F protein (RSV-preFΔCT). As preF is non-functional, RSV-preFΔCT was amplified in a production line expressing a functional substitute, and exhibited a single-cycle replication phenotype, which holds several unique potential advantages. RSV-preFΔCT prevented shedding and lung pathology after viral challenge in mice, but induced low levels of anti-attachment protein (G) antibodies (Abs). Given the significant contributions of anti-G Abs toward disease prevention, we generated modifications to RSV-preFΔCT in an effort to induce higher anti-G Ab levels. The Ab levels were monitored after the prime-boost vaccination of mice with modified vaccines. The most successful modification for enhancing induced anti-G Abs was seen with the placement of G in the first genome position. This vaccine also reduced the pathology after challenge with a high dose of wt RSV, and outperformed the sera from wt RSV-vaccinated mice in in vitro neutralization. Thus, raising the anti-G Ab levels induced by RSV-preFΔCT enhanced efficacy in vitro and in vivo, and constitutes an important next step in developing a live, single-cycle, efficacious vaccine for the human population.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Humanos , Camundongos , Animais , Proteínas Virais de Fusão/genética , Anticorpos Neutralizantes , Anticorpos Antivirais , Vírus Sincicial Respiratório Humano/genéticaRESUMO
Live-attenuated Respiratory syncytial virus (RSV) vaccines given intranasally have potential to provide comprehensive protection, including lung-resident immunity. It has however proven challenging to impart both sufficient safety and efficacy in a vaccine. To achieve the latter, we used a trans-complementing approach to generate live single-cycle RSV vaccines expressing the prefusion form (preF) of the viral fusion protein (F), either membrane-anchored or secreted. Both viruses were tested for their ability to induce a protective immune response in mice after intranasal prime-boost vaccination. The secreted preF vaccine failed to induce a protective response. The anchored preF vaccine induced anti-preF antibodies and antiviral T cells, and protected mice from lung pathology and viral shedding after challenge. Neither vaccine induced anti-G antibodies, for reasons unknown. In spite of the latter and single-cycle replication, the membrane-anchored preF vaccine was protective and demonstrates potential for development of an efficacious live vaccine with a stable safety phenotype.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Camundongos , Animais , Vacinas contra Vírus Sincicial Respiratório/genética , Vírus Sincicial Respiratório Humano/genética , Anticorpos Antivirais , Anticorpos Neutralizantes , Proteínas Virais de Fusão/genéticaRESUMO
Respiratory syncytial virus (RSV) is the major viral respiratory pathogen for human infants and children. Despite a severe global burden incurred by annual RSV epidemics, there is no licensed RSV vaccine. We have developed an RSV vaccine from a human RSV strain from which the gene for the viral M protein has been deleted ("Mnull RSV"). RSV infects airway cells and produces each of its proteins. The M protein is responsible for reassembling the various other synthesized viral proteins into new, intact virus. In the absence of the M protein, therefore, reassembly does not occur, and the Mnull RSV does not replicate. We vaccinated 2-week old infant baboons with Mnull RSV either intranasally (IN) or directly into the lung (intratracheal, or IT), then infected these animals by inoculating human RSV directly into the lung. IN vaccination induced inconsistent serum RSV neutralizing antibody (NA) responses, but provided moderate reductions in respiratory rates, overall signs of illness and viral replication in bronchoalveolar lavage (BAL) fluid following infection. Intratracheal vaccination induced much stronger RSV NA responses, which persisted for at least 4-6â¯months. Following RSV infection, animals vaccinated by the IT route had much greater reductions in tachypnea and work of breathing than animals vaccinated IN, and had undetectable amounts of virus in BAL fluids. These results support the further development of IT Mnull RSV vaccination to reduce the impact of RSV infection in humans.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Criança , Humanos , Lactente , Papio , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinação , Replicação ViralRESUMO
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 de 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 Antigênicas/efeitos adversos , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Human respiratory syncytial virus (HRSV) is released from the apical membrane of polarized epithelial cells. However, little is known about the processes of assembly and release of HRSV and which viral gene products are involved in the directional maturation of the virus. Based on previous studies showing that the fusion (F) glycoprotein contained an intrinsic apical sorting signal and that N- and O-linked glycans can act as apical targeting signals, we investigated whether the glycoproteins of HRSV were involved in its directional targeting and release. We generated recombinant viruses with each of the three glycoprotein genes deleted individually or in groups. Each deleted gene was replaced with a reporter gene to maintain wild-type levels of gene expression. The effects of deleting the glycoprotein genes on apical maturation and on targeting of individual proteins in polarized epithelial cells were examined by using biological, biochemical, and microscopic assays. The results of these studies showed that the HRSV glycoproteins are not required for apical maturation or release of the virus. Further, deletion of one or more of the glycoprotein genes did not affect the intracellular targeting of the remaining viral glycoproteins or the nucleocapsid protein to the apical membrane.
Assuntos
Polaridade Celular , Células Epiteliais/metabolismo , Vírus Sincicial Respiratório Humano/fisiologia , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular , Chlorocebus aethiops , DNA Complementar , Cães , Deleção de Genes , Genes Reporter , Engenharia Genética , Glicoproteínas/análise , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Rim/citologia , Neoplasias Hepáticas/patologia , Fases de Leitura Aberta , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/patogenicidade , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Perceived inefficiency and inadequate knowledge of the human respiratory syncytial virus (hRSV) assembly process present a hurdle for large-scale production of authentic hRSV virus-like particles (VLPs) for vaccine purposes. We previously established that the matrix protein, phosphoprotein (P), and fusion protein carboxy-terminus were sufficient to generate VLPs that resemble filamentous wildtype hRSV. Here, the contribution of P was examined. By co-expressing matrix, fusion, and modified P proteins, a ser/thr-rich P region (residues 39-57) was found to be critical for VLP formation, whereas the oligomerization domain was not. Substitutions throughout region 39-57 inhibited VLP formation and relevant amino acids were identified. Phosphomimetic substitutions of serines and threonines inhibited VLP formation; Phosphoblatant substitutions did not. The data show that P not only co-regulates replication and transcription but also has an important role in assembly, mediated by a separate domain that likely interacts with M and/or F and is highly regulated by phosphorylation.
Assuntos
Regulação Viral da Expressão Gênica , Fosfoproteínas/genética , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais de Fusão/genética , Proteínas da Matriz Viral/genética , Vírion/genética , Substituição de Aminoácidos , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Plasmídeos/química , Plasmídeos/metabolismo , Domínios Proteicos , Vacinas contra Vírus Sincicial Respiratório/biossíntese , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/metabolismo , Transfecção , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Vírion/crescimento & desenvolvimento , Vírion/metabolismoRESUMO
Current efforts to develop a vaccine against human respiratory syncytial virus (HRSV) are focused on live attenuated strains. However, the unstable nature of HRSV is a major challenge for the preparation, storage and distribution of live vaccine candidates. We report here that the stability of HRSV can be improved by incorporation of the GP64 glycoprotein from baculovirus Autographa californica multiple nucleopolyhedrovirus. GP64 was incorporated in place of or in addition to the homologous HRSV glycoproteins and was either expressed from the HRSV genome or provided by propagating the virus in a Vero cell line constitutively expressing GP64 (Vbac cells). The infectivity of the different virus stocks was monitored after storage at 4 degrees, 22 degrees or 37 degrees C, over a period of 8 weeks. The results showed that the infectivity of HRSV could be stabilized by up to 10,000-fold by the GP64 protein, when stored at 22 degrees C for 6 weeks. This approach for stabilizing live HRSV may be important for vaccine development and may also prove useful for stabilizing other enveloped viruses.
Assuntos
Moléculas de Adesão Celular/genética , Glicoproteínas de Membrana/genética , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais/genética , Animais , Baculoviridae/genética , Baculoviridae/imunologia , Western Blotting , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Engenharia Genética , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/patogenicidade , Temperatura , Células VeroRESUMO
The importance of the F protein cytoplasmic tail (CT) for replication of human respiratory syncytial virus (HRSV) was examined by monitoring the behavior of viruses expressing F proteins with a modified COOH terminus. The F protein mutant viruses were recovered and amplified under conditions where F protein function was complemented by expression of a heterologous viral envelope protein. The effect of the F protein modifications was then examined in the context of a viral infection in standard cell types (Vero and HEp-2). The F protein modifications consisted of a deletion of the predicted CT or a replacement of the CT with the CT of the vesicular stomatitis virus (VSV) G protein. In addition, engineered HRSVs that lacked all homologous glycoprotein genes (SH, G, and F) and expressed instead either the authentic VSV G protein or a VSV G containing the HRSV F protein CT were examined. We found that deletion or replacement of the F protein CT seriously impaired the production of infectious progeny. Cells infected with viruses bearing CT modifications displayed increased F protein surface expression and increased syncytium formation. The distribution of F protein in the plasma membrane of infected cells was altered, resulting in an F protein that was evenly distributed rather than localized predominantly to virus-induced surface filaments. CT deletion or exchange also abrogated interaction of F protein with Triton-insoluble lipid rafts. Addition of the F protein CT to the VSV G protein, expressed as the only viral glycoprotein in an HRSV genome, had the opposite effects: the number of infectious progeny was higher, the surface distribution was changed from relatively even to localized, and the proportion of VSV G protein associated with lipid rafts was higher. Together, these results show that the HRSV F protein CT plays a critical role in F protein cellular localization and production of infectious virus and suggest that the function provided by the CT is independent of the F protein ectodomain and transmembrane domain and is mediated by F protein-lipid raft interaction.