No 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. IMPORTANCENovel 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. Influenza A and B viruses remain a substantial public health burden, with seasonal epidemics resulting in significant morbidity, mortality, and economic loss (1-3). Pandemic outbreaks occur when antigenically novel influenza A viruses emerge in a population with little preexisting immunity (4). Pandemic viruses spread more rapidly and cause more severe di...

A cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators. The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Specifically, a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could potentially be used as adjuvants or immune modulators.

WNT signaling is involved in maintaining stem cells in an undifferentiated state; however, it is often unclear which WNTs and WNT receptors are mediating these activities. Here we examined the role of the WNT receptor FZD7 in maintaining human embryonic stem cells (hESCs) in an undifferentiated and pluripotent state. FZD7 expression is significantly elevated in undifferentiated cells relative to differentiated cell populations, and interfering with its expression or function, either by short hairpin RNA-mediated knockdown or with a fragment antigen binding (Fab) molecule directed against FZD7, disrupts the pluripotent state of hESCs. The FZD7-specific Fab blocks signaling by Wnt3a protein by downregulating FZD7 protein levels, suggesting that FZD7 transduces Wnt signals to activate Wnt/β-catenin signaling. These results demonstrate that FZD7 encodes a regulator of the pluripotent state and that hESCs require endogenous WNT/β-catenin signaling through FZD7 to maintain an undifferentiated phenotype. human pluripotent stem cell | self-renewal | differentiation