Respiratory Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form Protein Nanoparticles That Induce Protective Immunity in Cotton Rats

PLOS ONE, Dec 2019

Respiratory Syncytial Virus (RSV) is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F) surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

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Respiratory Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form Protein Nanoparticles That Induce Protective Immunity in Cotton Rats

et al. (2012) Respiratory Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form Protein Nanoparticles That Induce Protective Immunity in Cotton Rats. PLoS ONE 7(11): e50852. doi:10.1371/journal.pone.0050852 Respiratory Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form Protein Nanoparticles That Induce Protective Immunity in Cotton Rats Gale Smith 0 Rama Raghunandan 0 Yingyun Wu 0 Ye Liu 0 Michael Massare 0 Margret Nathan 0 Bin Zhou 0 Hanxin Lu 0 Sarathi Boddapati 0 Jingning Li 0 David Flyer 0 Gregory Glenn 0 Steven M. Varga, University of Iowa, United States of America 0 1 Discovery , Novavax Incorporated, Rockville, Maryland , United States of America, 2 Process Development , Novavax Incorporated, Rockville, Maryland , United States of America, 3 Formulation Development , Novavax Incorporated, Rockville, Maryland , United States of America, 4 Analytical Development , Novavax Incorporated, Rockville, Maryland , United States of America, 5 Clinical Development , Novavax Incorporated, Rockville, Maryland , United States of America Respiratory Syncytial Virus (RSV) is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F) surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate. - Competing Interests: The authors have reviewed the PLOS ONE guidelines for authors and declare: Each of the authors are employed by Novavax, Inc., Rockville, MD, a public biotechnology company, and GS and MM hold patents owned by Novavax. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory infection in infants and young children, and a major disease burden in the elderly. Despite the fact that the RSV virus was characterized half a century ago, there is currently no vaccine for RSV and development has been hampered by vaccine-mediated disease enhancement in children administered a formalin inactivated RSV in the 1960s [1,2]. Challenges in antigen production, purity, stability, and potency of RSV vaccine candidates have also been impediments to development [35]. The RSV fusion glycoprotein (F) mediates viral entry into cells and cell to cell fusion, is a target of neutralizing antibodies, and highly conserved between RSV A and B strains [6,7]. RSV F is produced as a precursor (F0) that is cleaved at Arg109 and Arg136 by cellular furin to three fragments, a shorter F2 polypeptide at the N-terminus covalently linked by two disulfides to a longer F1 polypeptide with an 18 amino acid fusion domain at the N-terminus and a hydrophobic membrane spanning region near the C-terminus; the intervening 27 amino acid fragment is released. Neutralizing monoclonal antibodies palivizumab and motavizumab bind to RSV F antigenic site II (Asn258 - Val278) [8] and have been shown to protect against both lower and upper respiratory RSV disease in high risk and term infants [9,10] The structures of the RSV F epitope polypeptides that bind these neutralizing antibodies are larger than the linear peptide and palivizumab binds with nanomolar and motavizumab picomolar affinity to RSV F [1113]. Modeling predicts that the full extent of the binding of palivizumab and motavizumab requires amino acids from one or two RSV F protomers, respectively. Therefore preserving RSV F tertiary and quaternary structures may be important in the development of an RSV F vaccine to preserve the native conformation of this important neutralizing region. In this report an oligomeric form of a modified full length RSV F was efficiently produced in Sf9 insect cells using a baculovirus vector. Recombinant RSV F extracted from cellular membranes and purified, assembled into nanoparticles with morphology c (...truncated)


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Gale Smith, Rama Raghunandan, Yingyun Wu, Ye Liu, Michael Massare, Margret Nathan, Bin Zhou, Hanxin Lu, Sarathi Boddapati, Jingning Li, David Flyer, Gregory Glenn. Respiratory Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form Protein Nanoparticles That Induce Protective Immunity in Cotton Rats, PLOS ONE, 2012, Volume 7, Issue 11, DOI: 10.1371/journal.pone.0050852