scholarly journals Obtaining and Characterization of the Monoclonal Antibodies Against G-Protein of the Respiratory Syncytial Virus

2020 ◽  
pp. 7-14
Author(s):  
N. A. Demidova ◽  
R. R. Klimova ◽  
A. A. Kushch ◽  
E. I. Lesnova ◽  
O. V. Masalova ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rsv Infection ◽  
Hybridoma Technology ◽  
Infected Cells ◽  
Syncytial Virus

The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.

scholarly journals Obtaining and Characterization of the Monoclonal Antibodies Against G-Protein of the Respiratory Syncytial Virus

2020 ◽  
Vol 97 (1) ◽  
pp. 7-14
Author(s):  
N. A. Demidova ◽  
R. R. Klimova ◽  
A. A. Kushch ◽  
E. I. Lesnova ◽  
O. V. Masalova ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rsv Infection ◽  
Hybridoma Technology ◽  
Infected Cells ◽  
Syncytial Virus

The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.

scholarly journals Heparin-Like Structures on Respiratory Syncytial Virus Are Involved in Its Infectivity In Vitro

1998 ◽  
Vol 72 (9) ◽  
pp. 7221-7227 ◽  
Author(s):  
C. Bourgeois ◽  
J. B. Bour ◽  
K. Lidholt ◽  
C. Gauthray ◽  
P. Pothier
Keyword(s):  
Respiratory Syncytial Virus ◽  
Heparan Sulfate ◽  
G Protein ◽  
Enzymatic Digestion ◽  
Virus Infectivity ◽  
Rsv Infection ◽  
Infected Cells ◽  
Virus Culture ◽  
Syncytial Virus

ABSTRACT Addition of heparin to the virus culture inhibited syncytial plaque formation due to respiratory syncytial virus (RSV). Moreover, pretreatment of the virus with heparinase or an inhibitor of heparin, protamine, greatly reduced virus infectivity. Two anti-heparan sulfate antibodies stained RSV-infected cells, but not noninfected cells, by immunofluorescence. One of the antibodies was capable of neutralizing RSV infection in vitro. These results prove that heparin-like structures identified on RSV play a major role in early stages of infection. The RSV G protein is the attachment protein. Both anti-heparan sulfate antibodies specifically bound to this protein. Enzymatic digestion of polysaccharides in the G protein reduced the binding, which indicates that heparin-like structures are on the G protein. Such oligosaccharides may therefore participate in the attachment of the virus.

scholarly journals Prophylactic Administration of a Complementarity-Determining Region Derived from a Neutralizing Monoclonal Antibody Is Effective against Respiratory Syncytial Virus Infection in BALB/c Mice

1998 ◽  
Vol 72 (1) ◽  
pp. 807-810 ◽  
Author(s):  
C. Bourgeois ◽  
J. B. Bour ◽  
L. S. Aho ◽  
P. Pothier
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Respiratory Syncytial Virus ◽  
Lung Infection ◽  
Neutralizing Monoclonal Antibody ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Complementarity Determining Region ◽  
Single Peptide

ABSTRACT Immunotherapy with antibodies against respiratory syncytial virus (RSV) is a treatment option given the absence of any vaccine or other available satisfactory treatment. We selected one of our monoclonal antibodies, RS-348, that is highly neutralizing. We showed that a single peptide (PEP3H) derived from complementarity-determining region 3 (CDR3) of its heavy chain was capable of neutralizing the virusin vitro. When intranasally administered 24 h before challenge, this peptide protected BALB/c mice against RSV lung infection. These results indicate that a single CDR can be effective against RSV infection.

scholarly journals DEVELOPMENT AND PROPERTIES OF NEUTRALIZING MONOCLONAL ANTIBODIES FOR FUSION PROTEIN OF RESPIRATORY SYNCYTIAL VIRUS

2019 ◽  
Vol 64 (2) ◽  
pp. 90-96
Author(s):  
A. A. Kushch ◽  
R. R. Klimova ◽  
N. E. Fedorova ◽  
O. V. Masalova ◽  
A. A. Niconova ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Respiratory Syncytial Virus ◽  
Culture Method ◽  
The Elderly ◽  
F Protein ◽  
Wide Range ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Tract Infections

Introduction. Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants and the elderly. The absence of a wide range of therapeutic drugs and vaccines indicates to the high relevance of the development of new effective drugs for the prevention and treatment of RSV infections. Purpose: to obtain highly active and specific monoclonal antibodies (MAbs) capable of detecting RSV in infected cells and neutralizing the infectious activity of the virus in vitro. Material and methods. RSV reference strains of group A 2 subgroups (A2 and Long) were propagated in HEp-2 and MA-104 cell lines, respectively. Mice were immunized with purified RSV A2 virus. MAbs were obtained using hybridoma technology. Results. A panel of 6 MAbs reacting with RSV strains А2 and Long has been obtained. Four MAbs were IgG (IgG2a or IgG2b subtype), two MAbs were IgM. All MAbs reacted with RSV F-protein in immunochemical tests. The MAbs actively reacted with RSV in ELISA, in immufluorescence and peroxidase staining of infected cells, and in immunodot test. Five out of 6 MAbs neutralized of RSV in cell culture. Different properties of MAbs suggest that they target different antigenic sites of F-protein. Discussion. Comparative analysis suggests that the obtained MAbs can be used for the development of diagnostic preparations, for RSV detection in clinical materials and confirmation of infection etiology by rapid culture method. Conclusion. High activity and specificity of MAbs indicate that they can serve as a basis for development vaccines and preventive medicines.

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

scholarly journals Inhibitory Effect of Sargassum fusiforme and Its Components on Replication of Respiratory Syncytial Virus In Vitro and In Vivo

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 548
Author(s):  
Kiramage Chathuranga ◽  
Asela Weerawardhana ◽  
Niranjan Dodantenna ◽  
Lakmal Ranathunga ◽  
Won-Kyung Cho ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antiviral Agent ◽  
Active Components ◽  
Sargassum Fusiforme ◽  
Antiviral Effects ◽  
Improve Life Expectancy ◽  
Rsv Infection ◽  
Syncytial Virus

Sargassum fusiforme, a plant used as a medicine and food, is regarded as a marine vegetable and health supplement to improve life expectancy. Here, we demonstrate that S. fusiforme extract (SFE) has antiviral effects against respiratory syncytial virus (RSV) in vitro and in vivo mouse model. Treatment of HEp2 cells with a non-cytotoxic concentration of SFE significantly reduced RSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and syncytium formation. Moreover, oral inoculation of SFE significantly improved RSV clearance from the lungs of BALB/c mice. Interestingly, the phenolic compounds eicosane, docosane, and tetracosane were identified as active components of SFE. Treatment with a non-cytotoxic concentration of these three components elicited similar antiviral effects against RSV infection as SFE in vitro. Together, these results suggest that SFE and its potential components are a promising natural antiviral agent candidate against RSV infection.

scholarly journals 667. Preclinical Pharmacokinetic and Pharmacodynamic Characterization of EDP-938, a Novel and Potent NonFusion Replication Inhibitor of Respiratory Syncytial Virus

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S305-S306
Author(s):  
Li-Juan Jiang ◽  
Lisha Xu ◽  
Meng Huang ◽  
Shucha Zhang ◽  
Yang Li ◽  
...  
Keyword(s):  
Viral Load ◽  
Respiratory Syncytial Virus ◽  
Post Infection ◽  
Human Liver ◽  
Liver Microsomes ◽  
Monkey Model ◽  
Green Monkey ◽  
Rsv Infection ◽  
Syncytial Virus

Abstract Background Respiratory syncytial virus (RSV) infection presents a significant health challenge in young children, elderly and immunocompromised patients. To date, there are no effective treatments available. EDP-938 was designed to meet this unmet medical need and is currently in Phase 2 clinical trials. Herein we report its preclinical pharmacokinetic (PK) and pharmacodynamic (PD) properties. Methods The pharmacokinetics of EDP-938 following single intravenous and oral doses were determined in mice, rats, dogs, and monkeys. In vitro cellular permeability and metabolic stability were assayed using Caco-2 cells and human liver microsomes, respectively. In vivo pharmacodynamic efficacy of EDP-938 was conducted in the African green monkey model, in which animals experimentally challenged with RSV were orally dosed twice daily with 100 mg/kg EDP-938 for 6 days starting 24 hours prior to infection. Results EDP-938 was well absorbed in the preclinical species with oral bioavailability values ranging from 27.1% in dogs, 35.4% in mice, 35.7% in rats, and 39.5% in monkeys, after a single oral dose when formulated in 0.5% methylcellulose. EDP-938 showed a moderate in vitro permeability of 3.6 x 10–6 cm/sec in Caco-2 cells. Based on the outcome of these absorption studies, EDP-938 was projected to have good oral absorption in humans. EDP-938 had low intrinsic clearance of 5 mL/minute/mg in human liver microsomes. Moreover, EDP-938 demonstrated potent antiviral efficacy in an African green monkey model of RSV infection. In untreated monkeys the RSV RNA viral load in the bronchoalveolar lavage fluid peaked at 106 copies/mL on day 5 post-infection, by comparison in animals treated with EDP-938 the viral load was below the limit of detection by day 3 post-infection. The PK/PD modeling suggested that plasma trough concentrations ≥10 × EC90 led to >4-log viral load reduction in EDP-938 treated monkeys. Conclusion The favorable preclinical PK and PD properties of EDP-938 support its further clinical development as a novel treatment for RSV infection. Disclosures All authors: No reported disclosures.

scholarly journals The Human Immune Response to Respiratory Syncytial Virus Infection

2017 ◽  
Vol 30 (2) ◽  
pp. 481-502 ◽  
Author(s):  
Clark D. Russell ◽  
Stefan A. Unger ◽  
Marc Walton ◽  
Jürgen Schwarze
Keyword(s):  
Immune Response ◽  
Respiratory Syncytial Virus ◽  
T Cell ◽  
T Cell Response ◽  
Rsv Infection ◽  
Human Immune Response ◽  
Ifn Γ ◽  
Human Immune ◽  
Syncytial Virus

SUMMARY Respiratory syncytial virus (RSV) is an important etiological agent of respiratory infections, particularly in children. Much information regarding the immune response to RSV comes from animal models and in vitro studies. Here, we provide a comprehensive description of the human immune response to RSV infection, based on a systematic literature review of research on infected humans. There is an initial strong neutrophil response to RSV infection in humans, which is positively correlated with disease severity and mediated by interleukin-8 (IL-8). Dendritic cells migrate to the lungs as the primary antigen-presenting cell. An initial systemic T-cell lymphopenia is followed by a pulmonary CD8+ T-cell response, mediating viral clearance. Humoral immunity to reinfection is incomplete, but RSV IgG and IgA are protective. B-cell-stimulating factors derived from airway epithelium play a major role in protective antibody generation. Gamma interferon (IFN-γ) has a strongly protective role, and a Th2-biased response may be deleterious. Other cytokines (particularly IL-17A), chemokines (particularly CCL-5 and CCL-3), and local innate immune factors (including cathelicidins and IFN-λ) contribute to pathogenesis. In summary, neutrophilic inflammation is incriminated as a harmful response, whereas CD8+ T cells and IFN-γ have protective roles. These may represent important therapeutic targets to modulate the immunopathogenesis of RSV infection.

scholarly journals Targeting the Respiratory Syncytial Virus N0-P Complex with Constrained α-Helical Peptides in Cells and Mice

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Marie Galloux ◽  
Nadège Gsponer ◽  
Vanessa Gaillard ◽  
Brice Fenner ◽  
Thibaut Larcher ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Small Molecules ◽  
Mechanisms Of Action ◽  
Rsv Infection ◽  
Macrocyclic Peptides ◽  
Escape Mutants ◽  
Severe Respiratory Infection ◽  
Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) is the main cause of severe respiratory infection in young children worldwide, and no therapies have been approved for the treatment of RSV infection. Data from recent clinical trials of fusion or L polymerase inhibitors for the treatment of RSV-infected patients revealed the emergence of escape mutants, highlighting the need for the discovery of inhibitors with novel mechanisms of action. Here we describe stapled peptides derived from the N terminus of the phosphoprotein (P) that act as replication inhibitors. We demonstrate that these peptides inhibit RSV replication in vitro and in vivo by preventing the formation of the N0-P complex. The present strategy provides a novel means of targeting RSV replication with constrained macrocyclic peptides or small molecules and is broadly applicable to other viruses of the Mononegavirales order.

scholarly journals Respiratory Syncytial Virus Infection Sensitizes Cells to Apoptosis Mediated by Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

2003 ◽  
Vol 77 (17) ◽  
pp. 9156-9172 ◽  
Author(s):  
Alexander Kotelkin ◽  
Elena A. Prikhod'ko ◽  
Jeffrey I. Cohen ◽  
Peter L. Collins ◽  
Alexander Bukreyev
Keyword(s):  
Respiratory Syncytial Virus ◽  
Tumor Necrosis Factor ◽  
Transcriptional Activation ◽  
Tumor Necrosis ◽  
Death Receptor ◽  
Rnase Protection ◽  
Rsv Infection ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Necrosis Factor

ABSTRACT Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease worldwide, especially in the pediatric population. For viruses in general, apoptotic death of infected cells is a mechanism for reducing virus replication. Apoptosis can also be an important factor in augmenting antigen presentation and the host immune response. We examined apoptosis in response to RSV infection of primary small airway cells, primary tracheal-bronchial cells, and A549 and HEp-2 cell lines. The primary cells and the A549 cell line gave generally similar responses, indicating their appropriateness as models in contrast to HEp-2 cells. With the use of RNase protection assays with probes representing 33 common apoptosis factors, we found strong transcriptional activation of both pro- and antiapoptotic factors in response to RSV infection, which were further studied at the protein level and by functional assays. In particular, RSV infection strongly up-regulated the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors death receptor 4 (DR4) and DR5. Furthermore, RSV-infected cells became highly sensitive to apoptosis induced by exogenous TRAIL. These findings suggest that RSV-infected cells in vivo are susceptible to killing through the TRAIL pathway by immune cells such as natural killer and CD4+ cells that bear membrane-bound TRAIL. RSV infection also induced several proapoptotic factors of the Bcl-2 family and caspases 3, 6, 7, 8, 9, and 10, representing both the death receptor- and mitochondrion-dependent apoptotic pathways. RSV also mediated the strong induction of antiapoptotic factors of the Bcl-2 family, especially Mcl-1, which might account for the delayed induction of apoptosis in RSV-infected cells in the absence of exogenous induction of the TRAIL pathway.

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