We previously demonstrated that the combination of synthetic small-molecule 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 murine challenge models. Combining the TLR4 and TLR7 ligands balances Th1 and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. Here, we demonstrate that the protective response induced in mice by this combined adjuvant is dependent upon TLR4 and TLR7 signaling via myeloid differentiation primary response gene 88 (MyD88), indicating that the adjuvants function in vivo via their known receptors, with negligible off-target effects, to induce protective immunity. The combined adjuvant acts via MyD88 in both bone marrow-derived and non-bone marrow-derived radioresistant cells to induce hemagglutinin-specific antibodies and protect mice against influenza virus challenge. The protective efficacy generated by immunization with this adjuvant and recombinant hemagglutinin antigen is transferable with serum from immunized mice to recipient mice in a homologous, but not a heterologous, H1N1 viral challenge model. Depletion of CD4⁺ cells after an established humoral response in immunized mice does not impair protection from a homologous challenge; however, it does significantly impair recovery from a heterologous challenge virus, highlighting an important role for vaccine-induced CD4⁺ cells in cross-protective vaccine efficacy. The combination of the two TLR agonists allows for significant dose reductions of each component to achieve a level of protection equivalent to that afforded by either single agent at its full dose.

IMPORTANCE Development of novel adjuvants is needed to enhance immunogenicity to provide better protection from seasonal influenza virus infection and improve pandemic preparedness. We show here that several dose combinations of synthetic TLR4 and TLR7 ligands are potent adjuvants for recombinant influenza virus hemagglutinin antigen induction of humoral and cellular immunity against viral challenges. The components of the combined adjuvant work additively to enable both antigen and adjuvant dose sparing while retaining efficacy. Understanding an adjuvant's mechanism of action is a critical component for preclinical safety evaluation, and we demonstrate here that a combined TLR4 and TLR7 adjuvant signals via the appropriate receptors and the MyD88 adaptor protein. This novel adjuvant combination contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

我们以前证明合成的小分子Toll样受体4（TLR4）和TLR7配体的组合是重组流感病毒血凝素的有效佐剂，可诱导快速和持续的免疫力，从而对同源，异源和异亚型的流感病毒起到保护作用鼠类挑战模型。结合TLR4和TLR7配体可平衡Th1和Th2型免疫应答，从而使针对病毒血凝素的长寿细胞和中和体液免疫力提高。在这里，我们证明了这种组合佐剂在小鼠中诱导的保护性应答依赖于TLR4和TLR7信号转导，后者通过髓样分化初级应答基因88（MyD88）进行，表明佐剂在体内具有功能通过其已知的受体，具有微不足道的脱靶作用，以诱导保护性免疫。组合的佐剂通过MyD88在骨髓来源和非骨髓来源的放射抗性细胞中起作用，以诱导血凝素特异性抗体并保护小鼠免受流感病毒攻击。用这种佐剂和重组血凝素抗原免疫产生的保护功效可以在同源但非异源的H1N1病毒攻击模型中用血清从免疫小鼠转移到受体小鼠。在免疫小鼠中建立体液反应后，CD4 ⁺细胞的耗竭不会损害对同源攻击的保护；然而，它确实严重损害了异源攻击病毒的恢复，突出了疫苗诱导的CD4的重要作用。⁺细胞在交叉保护性疫苗中的功效。两种TLR激动剂的组合可显着降低每种成分的剂量，以达到与任何一种单剂全剂量所提供的保护水平相当的保护水平。

重要性需要开发新的佐剂来增强免疫原性，以提供更好的保护以抵抗季节性流感病毒感染并提高大流行的防范能力。我们在这里显示，合成的TLR4和TLR7配体的几种剂量组合是重组流感病毒血凝素抗原诱导体液和细胞抵抗病毒攻击的有效佐剂。组合的佐剂的成分可加在一起发挥作用，以使抗原和佐剂的剂量均能保留，同时保持功效。了解佐剂的作用机制是临床前安全性评估的关键组成部分，我们在这里证明，结合的TLR4和TLR7佐剂通过适当的受体和MyD88衔接子蛋白发出信号。