title: Evaluation of an intranasal virosomal vaccine against respiratory syncytial virus in mice: effect of TLR2 and NOD2 ligands on induction of systemic and mucosal immune responses.
abstract: BACKGROUND
  RSV infection remains a serious threat to newborns and the elderly. Currently, there is no vaccine available to prevent RSV infection. A mucosal RSV vaccine would be attractive as it could induce mucosal as well as systemic antibodies, capable of protecting both the upper and lower respiratory tract. Previously, we reported on a virosomal RSV vaccine for intramuscular injection with intrinsic adjuvant properties mediated by an incorporated lipophilic Toll-like receptor 2 (TLR2) ligand. However, it has not been investigated whether this virosomal RSV vaccine candidate would be suitable for use in mucosal immunization strategies and if additional incorporation of other innate receptor ligands, like NOD2-ligand, could further enhance the immunogenicity and protective efficacy of the vaccine.
  
  
  OBJECTIVE
  To explore if intranasal (IN) immunization with a virosomal RSV vaccine, supplemented with TLR2 and/or NOD2-ligands, is an effective strategy to induce RSV-specific immunity.
  
  
  METHODS
  We produced RSV-virosomes carrying TLR2 (Pam3CSK4) and/or NOD2 (L18-MDP) ligands. We tested the immunopotentiating properties of these virosomes in vitro, using TLR2- and/or NOD2-ligand-responsive murine and human cell lines, and in vivo by assessing induction of protective antibody and cellular responses upon IN immunization of BALB/c mice.
  
  
  RESULTS
  Incorporation of Pam3CSK4 and/or L18-MDP potentiates the capacity of virosomes to activate (antigen-presenting) cells in vitro, as demonstrated by NF-κB induction. In vivo, incorporation of Pam3CSK4 in virosomes boosted serum IgG antibody responses and mucosal antibody responses after IN immunization. While L18-MDP alone was ineffective, incorporation of L18-MDP in Pam3CSK4-carrying virosomes further boosted mucosal antibody responses. Finally, IN immunization with adjuvanted virosomes, particularly Pam3CSK4/L18-MDP-adjuvanted-virosomes, protected mice against infection with RSV, without priming for enhanced disease.
  
  
  CONCLUSIONS
  Mucosal immunization with RSV-virosomes, supplemented with incorporated TLR2- and/or NOD2-ligands, represents a promising approach to induce effective and safe RSV-specific immunity.
authors.affiliation: Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Shafique
authors.forename: Tjarko
authors.initials: T
authors.lastname: Meijerhof
authors.forename: Jan
authors.initials: J
authors.lastname: Wilschut
authors.forename: Aalzen
authors.initials: A
authors.lastname: de Haan
mesh_terms: D000281:Administration, Intranasal; D000818:Animals; D002460:Cell Line; D005260:Female; D006801:Humans; D018928:Immunity, Mucosal / Q000276:immunology*; D008024:Ligands; D008168:Lung / Q000276:immunology / Q000473:pathology / Q000821:virology; D051379:Mice; D053473:Nod2 Signaling Adaptor Protein / Q000378:metabolism*; D012136:Respiratory Syncytial Viruses / Q000276:immunology* / Q000502:physiology; D013045:Species Specificity; D051195:Toll-Like Receptor 2 / Q000378:metabolism*; D022721:Vaccines, Virosome / Q000009:adverse effects* / Q000276:immunology*
chemical_list: D008024:Ligands; D053473:Nod2 Signaling Adaptor Protein; C495945:Nod2 protein, mouse; C493486:Tlr2 protein, mouse; D051195:Toll-Like Receptor 2; D022721:Vaccines, Virosome