title: Synthetic Toll-like receptor 4 (TLR4) and TLR7 ligands as influenza virus vaccine adjuvants induce rapid, sustained, and broadly protective responses.
abstract: Current vaccines against influenza virus infection rely on the induction of neutralizing antibodies targeting the globular head of the viral hemagglutinin (HA). Protection against seasonal antigenic drift or sporadic pandemic outbreaks requires further vaccine development to induce cross-protective humoral responses, potentially to the more conserved HA stalk region. Here, we present a novel viral vaccine adjuvant comprised of two synthetic ligands for Toll-like receptor 4 (TLR4) and TLR7. 1Z105 is a substituted pyrimido[5,4-b]indole specific for the TLR4-MD2 complex, and 1V270 is a phospholipid-conjugated TLR7 agonist. Separately, 1Z105 induces rapid Th2-associated IgG1 responses, and 1V270 potently generates Th1 cellular immunity. 1Z105 and 1V270 in combination with recombinant HA from the A/Puerto Rico/8/1934 strain (rPR/8 HA) effectively induces rapid and sustained humoral immunity that is protective against lethal challenge with a homologous virus. More importantly, immunization with the combined adjuvant and rPR/8 HA, a commercially available split vaccine, or chimeric rHA antigens significantly improves protection against both heterologous and heterosubtypic challenge viruses. Heterosubtypic protection is associated with broadly reactive antibodies to HA stalk epitopes. Histological examination and cytokine profiling reveal that intramuscular (i.m.) administration of 1Z105 and 1V270 is less reactogenic than a squalene-based adjuvant, AddaVax. In summary, the combination of 1Z105 and 1V270 with a recombinant HA induces rapid, long-lasting, and balanced Th1- and Th2-type immunity; demonstrates efficacy in a variety of murine influenza virus vaccine models assaying homologous, heterologous, and heterosubtypic challenge viruses; and has an excellent safety profile.
  
  
  OBJECTIVE
  Novel adjuvants are needed to enhance immunogenicity and increase the protective breadth of influenza virus vaccines to reduce the seasonal disease burden and ensure pandemic preparedness. We show here that the combination of synthetic Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous, and heterosubtypic challenge models. Combining TLR4 and TLR7 ligands balances Th1- and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. The combined adjuvant has an attractive safety profile and the potential to augment seasonal-vaccine breadth, contribute to a broadly neutralizing universal vaccine formulation, and improve response time in an emerging pandemic.
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Goff
authors.affiliation: UCSD Moores Cancer Center, University of California, San Diego, La Jolla, California, USA.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Luis
authors.initials: L
authors.lastname: Martínez-Gil
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California, USA.
authors.forename: Maripat
authors.initials: M
authors.lastname: Corr
authors.affiliation: UCSD Moores Cancer Center, University of California, San Diego, La Jolla, California, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Crain
authors.affiliation: UCSD Moores Cancer Center, University of California, San Diego, La Jolla, California, USA.
authors.forename: Shiyin
authors.initials: S
authors.lastname: Yao
authors.affiliation: UCSD Moores Cancer Center, University of California, San Diego, La Jolla, California, USA.
authors.forename: Howard B
authors.initials: HB
authors.lastname: Cottam
authors.affiliation: UCSD Moores Cancer Center, University of California, San Diego, La Jolla, California, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Chan
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Irene
authors.initials: I
authors.lastname: Ramos
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Dirk
authors.initials: D
authors.lastname: Eggink
authors.affiliation: Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Mitra
authors.initials: M
authors.lastname: Heshmati
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Florian
authors.initials: F
authors.lastname: Krammer
authors.affiliation: UCSD Moores Cancer Center, University of California, San Diego, La Jolla, California, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Messer
authors.affiliation: UCSD Moores Cancer Center, University of California, San Diego, La Jolla, California, USA.
authors.forename: Minya
authors.initials: M
authors.lastname: Pu
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Ana
authors.initials: A
authors.lastname: Fernandez-Sesma
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA peter.palese@mssm.edu dcarson@ucsd.edu.
authors.forename: Peter
authors.initials: P
authors.lastname: Palese
authors.affiliation: UCSD Moores Cancer Center, University of California, San Diego, La Jolla, California, USA peter.palese@mssm.edu dcarson@ucsd.edu.
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Carson
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage* / Q000138:chemical synthesis; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D056738:Cross Protection; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000276:immunology*; D007252:Influenza Vaccines / Q000008:administration & dosage / Q000276:immunology*; D007251:Influenza, Human / Q000276:immunology* / Q000821:virology; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018417:Th1 Cells / Q000276:immunology; D018418:Th2 Cells / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000819:agonists* / Q000276:immunology; D051199:Toll-Like Receptor 7 / Q000819:agonists* / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D000914:Antibodies, Viral; D007252:Influenza Vaccines; D008024:Ligands; C495345:TLR4 protein, human; C495348:TLR7 protein, human; D051197:Toll-Like Receptor 4; D051199:Toll-Like Receptor 7