An Analysis of Linker-Dependent Effects on the APC Activation and In Vivo Immunogenicity of an R848-Conjugated Influenza Vaccine.

Subunit or inactivated vaccines comprise the majority of vaccines used against viral and bacterial pathogens. However, compared to their live/attenuated counterparts, these vaccines often demonstrate reduced immunogenicity, requiring multiple boosters and or adjuvants to elicit protective immune responses. For this reason, studies of adjuvants and the mechanism through which they can improve inactivated vaccine responses are critical for the development of vaccines with increased efficacy. Studies have shown that the direct conjugation of adjuvant to antigen promotes vaccine immunogenicity, with the advantage of both the adjuvant and antigen targeting the same cell. Using this strategy of direct linkage, we developed an inactivated influenza A (IAV) vaccine that is directly conjugated with the Toll-like receptor 7/8 agonist resiquimod (R848) through a heterobifunctional crosslinker. Previously, we showed that this vaccine resulted in improved protection and viral clearance in newborn nonhuman primates compared to a non-adjuvanted vaccine. We subsequently discovered that the choice of linker used to conjugate R848 to the virus alters the stimulatory activity of the vaccine, promoting increased maturation and proinflammatory cytokine production from DC differentiated in vitro. With this knowledge, we explored how the choice of crosslinker impacts the stimulatory activity of these vaccines. We found that the linker choice alters signaling through the NF-κB pathway in human monocyte-derived dendritic cells (moDCs). Further, we extended our analyses to in vivo differentiated APC present in human peripheral blood, replicating the linker-dependent differences found in in vitro differentiated cells. Finally, we demonstrated in a mouse model that the choice of linker impacts the amount of IAV-specific IgG antibody produced in response to vaccination. These data enhance our understanding of conjugation approaches for improving vaccine immunogenicity.

Th17-Derived Cytokines Synergistically Enhance IL-17C Production by the Colonic Epithelium.

Tightly regulated communication between the gastrointestinal epithelium and immune cells in the underlying lamina propria is critical for immune homeostasis and inflammation. IL-17C, produced by epithelial cells after exposure to inflammatory stimuli, facilitates cell-to-cell communication by promoting inflammatory responses in Th17 cells. In this study, we demonstrate that Th17-derived cytokines TNF-α, IL-17A, and IL-22 synergistically enhance IL-17C expression in both human-transformed colonic epithelial cell lines and primary non-inflammatory bowel disease colonic epithelial spheroids. This synergistic expression requires activation of the transcription factor NF-κB downstream of the TNF-α stimulus, evidenced by the reduction of IL-17C expression in the presence of an IκBα inhibitor. IL-17A and IL-22 enhance IL-17C expression through the activation of the transcription factor AP-1 in a p38 MAPK-dependent manner. Colonic spheroids derived from uninvolved epithelial of ulcerative colitis patients stimulated with TNF-α, IL-17A, and IL-22 show muted responses compared with non-inflammatory bowel disease spheroids, and inflamed spheroids yielded more IL-17C expression in the presence of TNF-α, and no response to IL-22 stimulation. Altogether, a role for IL-17C in activating Th17 cells combined with our findings of Th17-derived cytokine-driven synergy in the expression of IL-17C identifies a novel inflammatory amplification loop in the gastrointestinal tract between epithelial cells and Th17 cells.

An Adenovirus early region 4 deletion mutant induces G2/M arrest via ATM activation and reduces expression of the mitotic marker phosphorylated (ser10) histone 3.

Adenovirus (Ad) type 5 (Ad5) early region 4 (E4) proteins inhibit the DNA damage response (DDR) including activation of the DDR kinase ATM and its substrates, which can induce G2/M cell cycle arrest. Infection with Ad5 or the E4 deletion mutant H5dl1007 (1007) resulted in the accumulation of post G1 cells with > 2 N cellular DNA content. A greater fraction of cells with 4 N DNA content was observed in 1007 infections compared to Ad5; this population was dependent on activation of ATM. G2/M checkpoint kinases, phosphorylated Chk2 (pChk2), and phosphorylated Cdk1 (pCdk1) were upregulated in 1007 infections, and 1007 showed reduced levels of the mitosis marker phosphorylated (Ser10) histone 3 compared to Ad5. Our results show that E4 mutant activation of ATM induces G2/M arrest via activation of checkpoint kinases, thereby contributing to viral-mediated regulation of the cell cycle.

IL-17C in human mucosal immunity: More than just a middle child.

Interleukin-17C (IL-17C) is an understudied member of the IL-17 family of cytokines. Its synthesis is induced by both cytokines and pathogenic stimuli in a variety of cell types, most often expressed at mucosal and barrier surfaces. IL-17C expression is dysregulated in a variety of autoinflammatory and autoimmune diseases including inflammatory bowel disease, psoriasis, and atopic dermatitis, yet it is protective against bacterial infections of the gut, skin, and lungs. In this review we highlight studies on IL-17C regulation and its function at human mucosal surfaces. Understanding the relationship between IL-17C and autoinflammatory and autoimmune diseases of the mucosa and defining the beneficial and pathogenic functions of the cytokine in inflammatory responses are the first steps in determining the potential for IL-17C as a therapeutic target.