Retinal Microglial Activation Following Topical Application of Intracellular Toll-Like Receptor Ligands.

PURPOSE: We previously have reported that application of the intracellular toll-like receptor (TLR)-9 ligand CpG-ODN onto the injured corneal surface induces widespread inflammation within the eye, including the retina. We tested the hypothesis that topical application of two other intracellular TLR agonists, Poly I:C and R848, would cause retinal microglial activation and migration into the subretinal space. METHODS: C57BL/6J wild-type and Cx3cr1gfp/+ mice were anesthetized and received central corneal abrasions followed by topical application of Poly I:C (TLR3 agonist), R848 (TLR7/8 agonist), or CpG-ODN (TLR9 agonist). Eyes were imaged in vivo by using spectral-domain optical coherence tomography to assess and quantify vitreous cells and retinal edema. Tissues were processed for whole-mount immunofluorescence staining or gene expression studies. Microglial activation was determined by morphologic changes, major histocompatibility complex (MHC) class II reactivity, and migration to the subretinal space. Expression of proinflammatory cytokine gene IL-6, IL-1ß, IFN-γ, and MCP-1 in retinal tissues were analyzed. RESULTS: At 24 hours, topical treatment with CpG-ODN and R848, but not Poly I:C, led to altered microglial morphology. One week after CpG-ODN and R848-treatment, eyes exhibited vitritis and mild inner retinal edema, increased number of subretinal Iba-1+ cells, and an increase in MHC II+ cells in the neural retina. Proinflammatory cytokine genes were upregulated after R848 treatment, whereas in the CpG-ODN group, only IL-1ß and MCP-1 were significantly upregulated. Retinal microglial activation was not observed in the Poly I:C-treated group. CONCLUSIONS: Topical application of CpG-ODN and R848, but not Poly I:C, to the damaged corneal surface can cause activation and migration of retinal microglia.

Induction of mucosal and systemic immunity against respiratory syncytial virus by inactivated virus supplemented with TLR9 and NOD2 ligands.

Respiratory syncytial virus (RSV) infection is the most important viral cause of severe respiratory disease in infants and children worldwide and also forms a serious threat in the elderly. The development of RSV vaccine, however, has been hampered by the disastrous outcome of an earlier trial using an inactivated and parenterally administered RSV vaccine which did not confer protection but rather primed for enhanced disease upon natural infection. Mucosal administration does not seem to prime for enhanced disease, but non-replicating RSV antigen does not induce a strong mucosal immune response. We therefore investigated if mucosal immunization with inactivated RSV supplemented with innate receptor ligands, TLR9 (CpG ODN) and NOD2 (L18-MDP) through the upper or total respiratory tract is an effective and safe approach to induce RSV-specific immunity. Our data show that beta-propiolactone (BPL) inactivated RSV (BPL-RSV) supplemented with CpG ODN and L18-MDP potentiates activation of antigen-presenting cells (APC) in vitro, as demonstrated by NF-κB induction in a model APC cell line. In vivo, BPL-RSV supplemented with CpG ODN/L18-MDP ligands induces local IgA responses and augments Th1-signature IgG2a subtype responses after total respiratory tract (TRT), but less efficient after upper respiratory tract (intranasal, IN) immunization. Addition of TLR9/NOD2 ligands to the inactivated RSV also promoted affinity maturation of RSV-specific IgG antibodies and shifted T cell responses from mainly IL-5-secreting cells to predominantly IFN-γ-producing cells, indicating a Th1-skewed response. This effect was seen for both IN and TRT immunization. Finally, BPL-RSV supplemented with TLR9/NOD2 ligands significantly improved the protection efficacy against a challenge with infectious virus, without stimulating enhanced disease as evidenced by lack of eotaxin mRNA expression and eosinophil infiltration in the lung. We conclude that mucosal immunization with inactivated RSV antigen supplemented with TLR9/NOD2 ligands is a promising approach to induce effective RSV-specific immunity without priming for enhanced disease.

The TLR9 ligand CpG promotes the acquisition of Plasmodium falciparum-specific memory B cells in malaria-naive individuals.

Despite the central role of memory B cells (MBC) in protective immune responses, little is understood about how they are acquired in naive individuals in response to Ag exposure, and how this process is influenced by concurrent activation of the innate immune system's TLR. In this longitudinal study of malaria-naive individuals, we examined the MBC response to two candidate malaria vaccines administered with or without CpG, a TLR9 ligand. We show that the acquisition of MBC is a dynamic process in which the vaccine-specific MBC pool rapidly expands and then contracts, and that CpG enhances the kinetics, magnitude, and longevity of this response. We observed that the percentage of vaccine-specific MBC present at the time of reimmunization predicts vaccine-specific Ab levels 14 days later; and that at steady-state, there is a positive correlation between vaccine-specific MBC and Ab levels. An examination of the total circulating MBC and plasma cell pools also suggests that MBC differentiate into plasma cells through polyclonal activation, independent of Ag specificity. These results provide important insights into the human MBC response, which can inform the development of vaccines against malaria and other pathogens that disrupt immunological memory.

Immunostimulatory DNA as a vaccine adjuvant.

Immunostimulatory DNA containing unmethylated CpG motifs is recognized by Toll-like receptor 9, resulting in the activation of innate immune responses that subsequently amplify the adaptive-immune response. Advances in the characterization of Toll-like receptor 9 signaling have identified immunostimulatory sequences (ISS) with distinct biological activities. Numerous animal models have demonstrated that synthetic ISS are effective adjuvants that enhance both humoral and cellular immune responses in diverse indications, ranging from infectious disease to cancer and allergy. An added benefit supporting the use of ISS as a vaccine adjuvant is that the specific activation of a pathway critical to the regulation of the immune response results in minimal toxicity. To date, clinical testing has largely affirmed the potency and safety of ISS-adjuvanted vaccines.