Glycoprotein Of Respiratory Syncytial Virus Research Articles

Outbreak of Nosocomial Respiratory Syncytial Virus Infections in a Hematology and Transplant Unit.

AbstractAbstract 3032Respiratory syncytial virus (RSV) is a pathogen associated with bronchiolitis in infants and small children. However, immunocompromised adults might also be at risk for severe lower respiratory tract involvement induced by RSV. We report the rapid control of an outbreak of nosocomial RSV infections in an institution with special focus on allogeneic and autologous stem cell transplantations.Between November 2011 and March 2012 56 patients were diagnosed with RSV infection by RT-PCR at our institution. In order to identify possible risk factors for lower respiratory tract involvement in RSV infected patients, clinical course of infection as well as radiological and laboratory findings were evaluated. Characterization of RSV strains in respiratory specimens was achieved by sequence analysis of part of the RSV glycoprotein G gene and phylogenetic comparison of outbreak and community strains was performed.Out of 56 patients with RSV infection, 39 patients showed signs of lower respiratory tract involvement. 14 fatal outcomes were observed. In most of the lethal cases coinfections with other pathogens such as Aspergillus spp. or Pseudomonas aeruginosa were present. Therefore the exact impact of RSV on the fatal outcome remains difficult, if not impossible, to assess. Age, sex, underlying disease, disease control or transplant status did not differ significantly between severe and non-severe cases. However, hypogammaglobulinemia was associated with a higher risk for fatal infection (p = 0.03) in univariate analysis, whereas therapy with oral ribavirin might show a protective effect (p = 0.02). Prolonged viral shedding was frequently observed (median 22.5 [1 – 143] days), most pronounced in patients folllowing allogeneic transplantation (p = 0.04). The major mode of RSV transmission seemed to be direct patient-to-patient contact as suggested by evaluation of patient movements on the wards. Sequence analysis on respiratory specimens obtained from 46 patients revealed the presence of a particular outbreak strain in 40 patients, characterized by an identical nucleotide sequence of RSV GA2 subtype. Phylogenetic analysis showed low prevalence of the outbreak strain in the community. By implementation of rigorous isolation measures containment of outbreak was achieved.Highly contagious viral pathogens such as RSV are able to cause rapidly spreading outbreaks among immunocompromised patients. Hypogammaglobulinemia might be a risk factor for severe lower respiratory tract infection. Treatment with oral ribavirin might have a positive effect on outcome. Rigorous isolation measures are essential to the containment of outbreak. Disclosures:Off Label Use: ribavirin aerosol is FDA approved for severe cases of RSV in infants and young children.

Long-Term and Memory Immune Responses in Mice against Newcastle Disease Virus-Like Particles Containing Respiratory Syncytial Virus Glycoprotein Ectodomains

Although respiratory syncytial virus (RSV) is a significant human pathogen, no RSV vaccines are available. We have reported that a virus-like particle (VLP) RSV vaccine candidate stimulated, in mice, robust, protective anti-RSV glycoprotein T(H)1 biased immune responses without enhanced respiratory disease upon RSV challenge. We report here an analysis of long-term responses to these VLPs. BALB/c mice immunized, without adjuvant, with VLPs or with infectious RSV generated anti-F and anti-G protein serum antibody responses that were stable over 14 months. Neutralizing antibody titers stimulated by VLPs were robust and durable for 14 months, whereas those of RSV-immunized animals declined significantly by 3 months. F protein-specific antibody-secreting cells were detected in the bone marrows of VLP-immunized mice but not in the marrows of RSV-immunized mice. Adoptive transfer of enriched splenic B cells from VLP-immunized mice into immunodeficient rag(-/-) mice resulted in anti-F and anti-G protein serum IgG antibody responses, in recipient mice, that were protective upon RSV challenge. In contrast, transfer of splenic B cells from RSV-immunized mice produced no detectable serum antibody in the recipients, nor could these mice inhibit RSV replication upon virus challenge. Immunization with VLPs stimulated the formation of germinal center GL7(+) B cells in normal mice. VLP immunization of TCR βδ(-/-) T-cell-deficient mice did not induce anti-RSV IgG antibodies, results consistent with T-cell-dependent immune responses. These results demonstrate that VLPs are effective in stimulating long-lived RSV-specific, T-cell-dependent neutralizing antibody-secreting cells and RSV-specific memory responses.

Dual Role of Respiratory Syncytial Virus Glycoprotein Fragment as a Mucosal Immunogen and Chemotactic Adjuvant

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.

Baculovirus-based Vaccine Displaying Respiratory Syncytial Virus Glycoprotein Induces Protective Immunity against RSV Infection without Vaccine-Enhanced Disease

BackgroundRespiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract diseases in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV yet. The attachment glycoprotein (G) of RSV is a potentially important target for protective antiviral immune responses. Recombinant baculovirus has been recently emerged as a new vaccine vector, since it has intrinsic immunostimulatory properties and good bio-safety profile.MethodsWe have constructed a recombinant baculovirus-based RSV vaccine, Bac-RSV/G, displaying G glycoprotein, and evaluated immunogenicity and protective efficacy by intranasal immunization of BALB/c mice with Bac-RSV/G.ResultsBac-RSV/G efficiently provides protective immunity against RSV challenge. Strong serum IgG and mucosal IgA responses were induced by intranasal immunization with Bac-RSV/G. In addition to humoral immunity, G-specific Th17- as well as Th1-type T-cell responses were detected in the lungs of Bac-RSV/G-immune mice upon RSV challenge. Neither lung eosinophilia nor vaccine-induced weight loss was observed upon Bac-RSV/G immunization and subsequent RSV infection.ConclusionOur data demonstrate that intranasal administration of baculovirus-based Bac-RSV/G vaccine is efficient for the induction of protection against RSV and represents a promising prophylactic vaccination regimen.

Developing Vaccines against Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV. The F and G glycoprotein of RSV are potentially important targets for protective antiviral immune responses. Here, recombinant replication-deficient adenovirus-based vaccines, rAd/3xG, expressing the soluble core domain of G glycoprotein (amino acids 131 to 230), engineered by codon optimization and tandem repetition for higher level expression, were constructed and evaluated for their potential as RSV vaccine in murine model. Strong serum IgG as well as mucosal IgA responses were induced by intranasal immunization of rAd/3xG. A single intranasal immunization of rAd/3xG vaccines provided potent protection against RSV challenge without vaccine-enhanced immunopathology. We also tested the efficacy of our vaccines against RSV field isolates from Korean pediatric patients and the data showed that our vaccine confers complete protection against RSV-A isolate challenge. We also show that sublingual or intranasal immunization of a procaryotically expressed and purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel selfadjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of our vaccine candidates elicit beneficial protective immunity and represent promising vaccine regimens preventing RSV infection.

Respiratory Syncytial Virus Glycoprotein G Interacts with DC-SIGN and L-SIGN To Activate ERK1 and ERK2

Respiratory syncytial virus (RSV) interaction with epithelial and dendritic cells (DCs) is known to require divalent cations, suggesting involvement of C-type lectins. RSV infection and maturation of primary human DCs are reduced in a dose-dependent manner by EDTA. Therefore, we asked whether RSV infection involves DC-SIGN (CD209) or its isoform L-SIGN (CD299) (DC-SIGN/R). Using surface plasmon resonance analysis, we demonstrated that the attachment G glycoprotein of RSV binds both DC- and L-SIGN. However, neutralization of DC- and L-SIGN on primary human DCs did not inhibit RSV infection, demonstrating that interactions between RSV G and DC- or L-SIGN are not required for productive infection. Thus, neither DC- nor L-SIGN represents a functional receptor for RSV. However, inhibition of these interactions increased DC activation, as evidenced by significantly higher levels of alpha interferon (IFN-α), MIP-1α, and MIP-1β in plasmacytoid DCs (pDCs) exposed to RSV after neutralization of DC-and L-SIGN. To understand the molecular interactions involved, intracellular signaling events triggered by purified RSV G glycoprotein were examined in DC- and L-SIGN-transfected 3T3 cells. RSV G interaction with DC- or L-SIGN was shown to stimulate ERK1 and ERK2 phosphorylation, with statistically significant increases relative to mock-infected cells. Neutralization of DC- and L-SIGN reduced ERK1/2 phosphorylation. With increased DC activation following DC- and L-SIGN neutralization and RSV exposure, these data demonstrate that the signaling events mediated by RSV G interactions with DC/L-SIGN are immunomodulatory and diminish DC activation, which may limit induction of RSV-specific immunity.

Virus Budding/Host Interactions

Viruses have developed unique and oftentimes complex molecular mechanisms to ensure efficient egress of mature virions from infected cells. Over the years, virologists have begun to unravel and appreciate the intricate roles of both viral and host proteins in this process, and particularly the specific recruitment of host factors to promote efficient budding of infectious virus. A better understanding of these virus-host interactions and the mechanisms of virus budding not only will provide fundamental insights into the functions of both viral and host proteins, but also will lead to the emergence of novel strategies to inhibit virion egress and spread. In this special issue on virus budding/host interactions, we have invited both review articles to summarize recent findings in the field as well as original research articles to provide new insights into this late stage of virus replication. The first paper of this special issue is a review article that focuses on the current knowledge of arenavirus budding and the critical role that the small RING finger Z protein plays in this process. S. Urata and J. C. de la Torre present a comprehensive discussion on the structure and function of Z and highlight the various L-domain motifs conserved in arenavirus Z proteins and in the matrix proteins of other emerging RNA viruses. Viral L-domains are of particular interest since they mediate recruitment of host proteins to promote virus budding and, as such, constitute attractive and potentially broad-spectrum targets for antiviral drugs designed to inhibit virus egress. Indeed, the authors discuss several strategies and ongoing efforts to identify and screen candidate budding inhibitors. The viral L-domain/host interaction theme continues in the second paper, where Y. Liu et al. have utilized a bimolecular complementation (BiMC) approach to detect, localize, and follow filovirus VP40-host complexes in live mammalian cells in real time. The authors postulate that adaptation of the BiMC approach to the study of virus-host interactions and budding may help address gaps in our understanding of the dynamics, kinetics, and trafficking patterns of virus-host complexes involved in the budding process. In addition, the BiMC approach was used in conjunction with the more established filovirus VP40 virus-like particle (VLP) budding assay to test candidate small molecule inhibitors (identified by in silico screening) of L-domain/host interactions for their ability to inhibit particle release. The mechanisms by which viral and host proteins are trafficked and/or targeted to the site of budding are of great interest. The third paper summarizes experimental data in support of a model for assembly and budding of influenza virus from viral bud zones and raft-enriched domains at the plasma membrane. Indeed, assembly and budding at raft domains appears to be a characteristic shared by several RNA virus families. M. Veit and B. Thaa discuss the raft-targeting features of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins of influenza virus, along with the critical roles of M1 and M2 proteins in orchestrating virus assembly and in membrane bending/particle scission, respectively. Little is known about the role of human respiratory syncytial virus (HRSV) glycoproteins during assembly and budding of mature virions. In the fourth paper, M. Batonick and G. W. Wertz address this gap by engineering recombinant viruses and using microscopic and biochemical analyses to demonstrate that deletion of the G and F proteins affected the incorporation of other viral proteins into budding virions; however, their absence did not directly affect the efficiency of virion egress. Thus, this study ascribes a novel role for the G and F glycoproteins during the late stage of HRSV replication. In the fifth paper, Snyder et al. describe a novel and intriguing cell lysis system utilized by two archaeal viruses, Sulfolobus turreted icosahedral virus (STIV) and Sulfolobus islandicus rod-shaped virus 2 (SIRV2). The authors demonstrate that the STIV c92 protein forms unique pyramid-like structures on the cell surface through which newly assembled virions are released during cell lysis. The authors speculate that this new lysis system may be common within other archaeal viral populations present in acidic hot springs. Ronald N. Harty Anthony P. Schmitt Fadila Bouamr Carolina B. Lopez Claude Krummenacher

Requirements for Human Respiratory Syncytial Virus Glycoproteins in Assembly and Egress from Infected Cells

Human respiratory syncytial virus (HRSV) is an enveloped RNA virus that assembles and buds from the plasma membrane of infected cells. The ribonucleoprotein complex (RNP) must associate with the viral matrix protein and glycoproteins to form newly infectious particles prior to budding. The viral proteins involved in HRSV assembly and egress are mostly unexplored. We investigated whether the glycoproteins of HRSV were involved in the late stages of viral replication by utilizing recombinant viruses where each individual glycoprotein gene was deleted and replaced with a reporter gene to maintain wild-type levels of gene expression. These engineered viruses allowed us to study the roles of the glycoproteins in assembly and budding in the context of infectious virus. Microscopy data showed that the F glycoprotein was involved in the localization of the glycoproteins with the other viral proteins at the plasma membrane. Biochemical analyses showed that deletion of the F and G proteins affected incorporation of the other viral proteins into budded virions. However, efficient viral release was unaffected by the deletion of any of the glycoproteins individually or in concert. These studies attribute a novel role to the F and G proteins in viral protein localization and assembly.

Conserved cysteine residues within the attachment G glycoprotein of respiratory syncytial virus play a critical role in the enhancement of cytotoxic T-lymphocyte responses.

The cytotoxic T-lymphocyte (CTL) response plays an important role in the control of respiratory syncytial virus (RSV) replication and the establishment of a Th1-CD4+ T cell response against the virus. Despite lacking Major Histocompatibility Complex I (MHC I)-restricted epitopes, the attachment G glycoprotein of RSV enhances CTL activity toward other RSV antigens, and this effect depends on its conserved central region. Here, we report that RSV-G can also improve CTL activity toward antigens from unrelated pathogens such as influenza, and that a mutant form of RSV-G lacking four conserved cysteine residues at positions 173, 176, 182, and 186 fails to enhance CTL responses. Our results indicate that these conserved residues are essential for the wide-spectrum pro-CTL activity displayed by the protein.

Surface display of respiratory syncytial virus glycoproteins in Lactococcus lactis NZ9000

A system for displaying heterologous respiratory syncytial virus (RSV) glycoproteins on the surface of Lactococcus lactis NZ9000 was developed. Fusion of the USP45 signal peptide and the cA (C terminus of the peptidoglycan-binding) domains of AcmA, a major autolysin from L. lactis, to the N- and C-terminal of the target proteins, respectively, was carried out. The target protein was the major immunogenic domain of either the F (40.17-kDa) or G (11.49-kDa) glycoprotein domains of the RSV. Whole-cell ELISA readings obtained after 24 h of induction showed an increase in protein expression as the cA domain repeats increased, for the G glycoprotein of RSV. On the other hand, the F glycoprotein indicated decreasing expression levels as the number of cA domain repeats increased. The difference in the expression levels of the F and G domains may be attributed to the different sizes of the antigenic domains. The size and properties of the target proteins are vital in determining the amount of antigenic domains being displayed on the surface of live cells. The system demonstrated here can aid in the utilization of the generally regarded as safe (GRAS) bacteria L. lactis, as a vaccine delivery vehicle to surface display the antigenic proteins of RSV.

A protein transduction domain located at the NH 2-terminus of human translationally controlled tumor protein for delivery of active molecules to cells

Protein transduction domains (PTDs) are small peptides, able to penetrate biological membranes and deliver various types of cargo both in vitro and in vivo. Because use of PTDs originating from viral origins resulted in undesired effects, PTDs originating from non-viral origins are needed. Here, we report that a 10-amino acid peptide (MIIYRDLISH) derived from the NH 2-terminus of human translationally controlled tumor protein (TCTP) functions as a PTD. This peptide was internalized through lipid raft-dependent endocytosis and partial macropinocytosis, and did not enter lysosome and nucleus. Beta-galactosidase fused to TCTP-PTD, when injected into mice, was efficiently delivered to liver, kidney, spleen, heart, and lungs of the animals. Preincubation of TCTP-PTD with adenovirus increased adenoviral mediated-gene expression in cells and also improved immune response to intranasally administered adenovirus expressing the triple repeat of G glycoprotein of respiratory syncytial virus (RSV), rAd/3×G. These findings suggest that TCTP-PTD might overcome the limitations of polycation-mediated transduction and serve as an efficient vehicle for drug delivery.

Evaluation of a synthetic peptide as a replacement for the recombinant fusion protein of respiratory syncytial virus in a potency ELISA

This report describes the development of a potency ELISA using a peptide derived from the motavizumab binding epitope of respiratory syncytial virus (RSV) F-protein. Motavizumab is an antibody therapeutic studied for the prevention of RSV disease. It binds to the RSV glycoprotein F (F-protein), blocking the ability of RSV to fuse with target cells. This binding is the basis for a potency ELISA, however, due to inefficient F-protein production, development of an alternative ligand for the potency ELISA was investigated. A series of synthetic peptides spanning the motavizumab epitope on F-protein were evaluated for motavizumab binding activity. A 26-mer peptide was identified with desirable motavizumab binding kinetics, as shown by ELISA and surface plasmon resonance. The peptide corresponds to a portion of the motavizumab binding domain on the F-protein, and is referred to as F-peptide. The binding of motavizumab to the F-peptide is used in a new motavizumab potency ELISA, which was shown to be robust and statistically comparable to the F-protein ELISA. In addition, based on a qualitative observation, this new ELISA may be able to detect motavizumab degradation with greater sensitivity compared to the F-protein ELISA.

Original Article: Molecular and demographic analysis of respiratory syncytial virus infection in patients admitted to King Chulalongkorn Memorial Hospital, Thailand, 2007

Please cite this paper as: Boonyasuppayakorn et al. (2010) Molecular and demographic analysis of respiratory syncytial virus infection in King Chulalongkorn Memorial Hospital admitted patients, Thailand, 2007. Influenza and Other Respiratory Viruses 4(5), 313–323.Objectives: To preliminary preview the molecular character and its possible clinical correlation of RSV subgroups in Thailand.Design: Cross‐sectional analytic design.Setting: Admitted acute lower respiratory tract infection patients of King Chulalongkorn Memorial Hospital, Bangkok, during Jun–Dec, 2007 were recruited.Sample: Nasopharyngeal aspirations were collected.Main outcome measures: All samples were analyzed for the presence of RSV glycoprotein G gene by reverse transcription PCR. Molecular character of each subgroup was determined by sequencing. Admission records were also analyzed for clinical correlations.Results: Equal infectivity and severity of both RSV subgroups to the patients was shown. Mixed infection was shown to be as common as each single infection, higher than previously reported. GA2 of subgroup A and BA‐IV of subgroup B were the most widespread genotypes and showed their monophyletic origins. From admission records, either type of infection did not show significantly preference in demographic record or clinical severity. Comorbidity, however, was statistically significant that more congenital heart disease was found in negative RSV cases, while more chronic pulmonary disease was in positive cases. Nevertheless, the clinical severity was insignificantly different suggesting that only patients with chronic pulmonary underlying were prone to be infected with RSV.Conclusions: This preliminary RSV study showed prevalence of subgroups, types of infection, and common genotypes in an epidemic, uncorrelated to demography or clinical severity.

Immune protection and mechanism of plasmid DNA encoding Gglycoprotein of respiratory syncytial virus(RSV)

Objective To construct a plasmid DNA encoding G glycoprotein of respiratory syncytial virus(RSV) and investigate the protective immune response against RSV infection. Methods Recombinant plasmid DNA of pcDNA3.1~G was constructed by standard RT-PCR based cloning procedure. The immunogenicity of recombinant G protein transiently expressed in HEK293 cells was detected by Western blot. BABL/c mice were intramuscularly immunized with pcDNA3.1~G. Samples of lung, sera, bronchoalveolar lavage fluid(BALF) were collected before and after RSV challenge; virus titer in lung was detected by viral titration; sections of paraffin embedding lung tissues were stained by haematoxylin and eosin(HE) for histological analyses; sera anti-RSV IgG levels were examined by ELISA; Th1/Th2 cytokine were detected by ELISA kit, the T lymphocyte subsets of BALF was determined by immunefluorescence staining followed by flow cytometry. Results Plasmid DNA of pcDNA3.1~G was successfully constructed. The expressed target protein possesses immunogenicity. After challenge, pcDNA3.1~G immunized mice presented relieved pathological changes in lung as well as reduced lung viral titers. The RSV specific IgG was detected in sera of immunized mice. There was significantly increased number of CD25~+CD4~+ T cells in mice BALF. Conclusion We constructed a pcDNA3.1~G plasmid DNA vaccination which can induce evident protective cellular immunity against RSV infection in mice with the increased number of CD25~+CD4~+ T cell subpopulation. Key words: Respiratory syncytial virus; G glycoprotein; Recombinant plasmid; T lymphocyte

The Role of Immunoprophylaxis in the Reduction of Disease Attributable to Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is an RNA virus that infects human respiratory epithelial cells and causes annual outbreaks of respiratory tract disease among infants and young children, as well as recurrent infections throughout life. Annual outbreaks of RSV disease are attributable to first-time infection in susceptible infants, reinfection in children and adults with waning or incomplete immunity, and infection by viral genotypes with sufficient antigenic variation to avoid innate and acquired immunity. In industrialized countries, few infectious diseases have a greater effect on the health of young children than does lower respiratory tract disease caused by RSV. By 2 years of age, almost all children will experience an RSV infection, and ∼50% will be infected twice.1 Results from the New Vaccine Surveillance Network (a prospective, population-based surveillance program sponsored by the Centers for Disease Control and Prevention [CDC]) define the burden of RSV disease in children younger than 5 years of age.2 An estimated 2 million children require medical care because of RSV infection, and ∼57500 children younger than 5 years are hospitalized annually. The major burden of RSV disease occurs among previously healthy infants and children whose susceptibility to severe RSV illness cannot be predicted by risk factors. Protection against RSV infection is mediated by serum antibody, secretory antibody, cytotoxic T lymphocytes, and innate immune responses. A vaccine offers the greatest promise for control of RSV disease, but vaccine development has been slowed by concerns about safety (enhancement of naturally occurring disease), the limited ability of infants to mount an immune response to RSV glycoprotein antigens, and the presence of maternal neutralizing antibody, which may attenuate an active immune response. Passive immunization with either a hyperimmune globulin or a monoclonal antibody preparation has been demonstrated in randomized, placebo-controlled trials to reduce the risk of hospitalization caused by … Address correspondence to H. Cody Meissner, MD, Tufts Medical Center, 750 Washington St, Boston, MA 02111. E-mail: cmeissner{at}tuftsmedicalcenter.org

Expression and purification of a secreted form of fusion glycoprotein of human respiratory syncytial virus encoded by recombinant baculovirus

To study the expression and purification of a secreted form of fusion glycoprotein (sF) of human respiratory syncytial virus (RSV) encoded by recombinant baculovirus. According the ORF of F protein, a pair of specific primers was designed and PCR technique was exploited to amplify the gene of sF in which the gene sequence of the transmembrane and cytoplasmic tail domains were replaced by a C-terminal six-histidine tag. Then, a recombinant baculovirus encoding sF-His was constructed, and transfected into sf9 insect cells by Lipofectamine cellfectine reagent. Finally, the expressed sF was purified by Ni2+ -affinity chromatograph. The gene encoding sF-His was obtained. The resulting construct of recombinant baculovirus is capable of expressing sF protein. The concentration of Ni2+ -affinity chromatograph purified sF is 1.084 mg/ml with the purity of no less than 90%. Baculovirus expression system is a good method for large scale of preparation of sF. The purified F paves the way for the development of potential RSV vaccine and diagnostic kit, etc.

Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.

Respiratory syncytial virus glycoproteins uptake occurs through clathrin-mediated endocytosis in a human epithelial cell line

Cell-surface viral proteins most frequently enter the cell through clathrin or caveolae endocytosis. Respiratory syncytial virus antigen internalization by immune cells is via caveolin, however, uptake of paramyxovirus cell membrane proteins by non-immune cells is done through clathrin-coated pits. In this work, the uptake of respiratory syncytial virus cell surface glycoproteins by non-immune human epithelial cells was investigated through indirect immunofluorescence with polyclonal anti-RSV antibody and confocal lasser-scanner microscopy. Clathrin and caveolae internalization pathways were monitored through specific inhibitors monodansylcadaverine (MDC) and methyl-beta-cyclodextrin (MBCD), respectively. Internalization of RSV antigens was inhibited by MDC but not by MBCD, implying that clathrin-mediated endocytosis is the major uptake route of RSV antigens by an epithelial human cell line.

Human Respiratory Syncytial Virus Glycoproteins Are Not Required for Apical Targeting and Release from Polarized Epithelial Cells

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.

Expression of chemokine receptor CX3CR1 in infants with respiratory syncytial virus bronchiolitis

Respiratory syncytial virus (RSV) glycoprotein G mimics fractalkine, a CX(3)C chemokine, which mediates chemotaxis of leukocytes expressing its receptor, CX(3)CR1. The aim of this study was to examine the relationship between RSV infection and expression of perforin and IFN-gamma in CX(3)CR1-expressing peripheral blood CD8(+) T cells. Samples were collected from infants with RSV bronchiolitis, both in the acute and convalescence phase (n = 12), and from their age- and sex-matched healthy controls (n = 15). Perforin expression and IFN-gamma secretion in CX(3)CR1(+) CD8(+) T cells were assessed by four-color flow cytometry. The NF-kappaB p50 and p65 subunit levels were also determined as markers of RSV-induced inflammation. Study results showed perforin and CX(3)CR1 expression to be significantly lower in the convalescent phase of infected infants than in healthy controls. There was no significant difference in IFN-gamma secretion and NF-kappaB binding activity between two time-points in RSV-infected infants, or when compared with healthy controls. Infants with prolonged wheezing had lower acute-phase CX(3)CR1 levels in peripheral blood. These data indicate existence of an event persisting after acute RSV infection that is able to modulate effector functions of cytotoxic T cells, and also link disease severity with CX(3)CR1 expression.