SUMMARY Systems biological analysis of immunity to the trivalent inactivated influenza vaccine (TIV) in humans revealed a correlation between early expression of TLR5 and the magnitude of the antibody response. Vaccination of Trl5−/− mice resulted in reduced antibody titers and lower frequencies of plasma cells, demonstrating a role for TLR5 in immunity to TIV. This was due to a failure to sense host microbiota. Thus, antibody responses in germ-free or antibiotic-treated mice were impaired, but restored by oral reconstitution with a flagellated, but not aflagellated, strain of E. coli. TLR5-mediated sensing of flagellin promoted plasma cell differentiation, directly, and by stimulating lymph node macrophages to produce plasma cell growth factors. Finally, TLR5-mediated sensing of the microbiota also impacted antibody responses to the inactivated polio vaccine, but not to adjuvanted vaccines or the live-attenuated yellow fever vaccine. These results reveal an unappreciated role for gut microbiota in promoting immunity to vaccination.

Conjugation of TLR agonists to protein or peptide antigens has been demonstrated in many studies to be an effective vaccine formula in inducing cellular immunity. However, the molecular and cellular mediators involved in TLR-induced immune responses have not been carefully examined. In this study, we identify Type I IFN and IL-12 as critical mediators of crosspriming induced by a TLR7 agonistantigen conjugate. We demonstrate that TLR7-driven cross-priming requires both Type I IFN and IL-12. Signaling through the IFN-␣␤R was required for the timely recruitment and accumulation of activated dendritic cells in the draining lymph nodes. Although IL-12 was indispensable during cross-priming, it did not regulate DC function. Therefore, the codependency for these 2 cytokines during TLR7-induced cross-priming is the result of their divergent effects on different celltypes. Furthermore, although dermal and CD8␣ ؉ DCs were able to cross-prime CD8 ؉ T cells, Langerhans cells were unexpectedly found to potently cross-present antigen and support CD8 ؉ T-cell expansion, both in vitro and in vivo. Collectively, the data show that a TLR7 agonistantigen conjugate elicits CD8 ؉ T-cell responses by the coordinated recruitment and activation of both tissue-derived and lymphoid organ-resident DC subsets through a Type I IFN and IL-12 codependent mechanism. (Blood. 2011;118(11): 3028-3038) IntroductionActivation of the innate immune system is a prerequisite to initiating adaptive immune responses. A major pathway eliciting these responses is the recognition of foreign bodies through Toll-like receptors (TLR), which results in the activation of antigen presenting cells (APC) and the production of a variety of proinflammatory mediators. 1 Previously, we showed that conjugation of a synthetic agonist targeting TLR7 to protein antigens results in a highly immunogenic vaccine that potently generates protective CD8 ϩ T-cell responses. 2 TLR7 is an intracellular receptor that recognizes single-stranded RNA molecules and detects RNA viruses such as the influenza virus. Stimulation of TLR7 has been shown in both mice and humans to result in vigorous production of multiple pro-inflammatory cytokines, including Type I IFN and IL-12. 3 The induction of Type I IFN and IL-12 is of particular interest given abundant evidence in the literature establishing these 2 cytokines as critical mediators of CD8 ϩ T-cell activation. 4,5 Type I IFN comprises a group of various IFN proteins, notably IFN␣ and IFN␤. IFNs are induced primarily during viral infections and have been shown to promote natural killer (NK), Type I helper T-cell (Th1), and CTL responses, 4 which are critical to combat viral infections through the elimination of virus-infected cells. Similarly, IL-12 also promotes the development of Th1 and CTL-mediated immunity. [6][7][8] However, the production of IL-12 is primarily associated with bacterial and parasitic infections. 7,9 The role of IL-12 during viral infections remains unclear as some reports indicate that CD8 ϩ T-cell responses elicit...

For more than a century immunologists and vaccinologists have existed in parallel universes. Immunologists have for long reveled in using “model antigens,” such as chicken egg ovalbumin or nitrophenyl haptens to study immune responses in model organisms such as mice. Such studies have yielded many seminal insights about the mechanisms of immune regulation, but their relevance to humans has been questioned. In another universe, vaccinologists have relied on human clinical trials to assess vaccine efficacy, but have done little to take advantage of such trials for studying the nature of immune responses to vaccination. The human model provides a nexus between these two universes, and recent studies have begun to use this model to study the molecular profile of innate and adaptive responses to vaccination. Such “systems vaccinology” studies are beginning to provide mechanistic insights about innate and adaptive immunity in humans. Here we present an overview of such studies, with particular examples from studies with the yellow fever and the seasonal influenza vaccines. Vaccination with the yellow fever vaccine causes a systemic acute viral infection and thus provides an attractive model to study innate and adaptive responses to a primary viral challenge. Vaccination with the live attenuated influenza vaccine causes a localized acute viral infection in mucosal tissues, and induces a recall response since most vaccinees have had prior exposure to influenza, and thus provides a unique opportunity to study innate and antigen-specific memory responses in mucosal tissues and in the blood. Vaccination with the inactivated influenza vaccine offers a model to study immune responses to an inactivated immunogen. Studies with these and other vaccines are beginning to reunite the estranged fields of immunology and vaccinology, and yielding unexpected insights about mechanisms of viral immunity. Therefore, vaccines that have been proven to be of immense benefit in saving lives offer us a new fringe benefit: lessons in viral immunology.

Covalent conjugation of TLR agonists to protein antigens often facilitates the generation of a CD8+ T cell response. However, mechanisms underlying the efficacy of the conjugate over its unconjugated counterpart have been largely uninvestigated. In this study, we show that conjugation of a TLR7 agonist enhances CD8+ T cell responses without affecting antigen persistence and with minimal impact on cellular uptake of the antigen in vivo. Instead, the conjugated form induced a robust accumulation of DCs in regional lymph nodes. Perhaps more importantly, cross-presentation in DCs was detected only when the antigen was delivered in the conjugated form with the TLR7 agonist. Collectively these data represent the first demonstration that a TLR agonist-antigen conjuagte elicits CD8+ T cell responses based not on its capacity to induce DC maturation or antigen persistence and uptake, but on the engagement of DC cross-presentation pathways.