Innate Molecular and Cellular Signature in the Skin Preceding Long-Lasting T Cell Responses after Electroporated DNA Vaccination.

DNA vaccines delivered with electroporation (EP) have shown promising results in preclinical models and are evaluated in clinical trials. In this study, we aim to characterize early mechanisms occurring in the skin after intradermal injection and EP of the auxoGTUmultiSIV DNA vaccine in nonhuman primates. First, we show that EP acts as an adjuvant by enhancing local inflammation, notably via granulocytes, monocytes/macrophages, and CD1aint-expressing cell recruitment. EP also induced Langerhans cell maturation, illustrated by CD86, CD83, and HLA-DR upregulation and their migration out of the epidermis. Second, we demonstrate the crucial role of the DNA vaccine in soluble factors release, such as MCP-1 or IL-15. Transcriptomic analysis showed that EP played a major role in gene expression changes postvaccination. However, the DNA vaccine is required to strongly upregulate several genes involved in inflammatory responses (e.g., Saa4), cell migration (e.g., Ccl3, Ccl5, or Cxcl10), APC activation (e.g., Cd86), and IFN-inducible genes (e.g., Ifit3, Ifit5, Irf7, Isg15, orMx1), illustrating an antiviral response signature. Also, AIM-2, a cytosolic DNA sensor, appeared to be strongly upregulated only in the presence of the DNA vaccine and trends to positively correlate with several IFN-inducible genes, suggesting the potential role of AIM-2 in vaccine sensing and the subsequent innate response activation leading to strong adaptive T cell responses. Overall, these results demonstrate that a combined stimulation of the immune response, in which EP and the auxoGTUmultiSIV vaccine triggered different components of the innate immunity, led to strong and persistent cellular recall responses.

A Single-Stranded Oligonucleotide Inhibits Toll-Like Receptor 3 Activation and Reduces Influenza A (H1N1) Infection.

The initiation of an immune response is dependent on the activation and maturation of dendritic cells after sensing pathogen associated molecular patterns by pattern recognition receptors. However, the response needs to be balanced as excessive pro-inflammatory cytokine production in response to viral or stress-induced pattern recognition receptor signaling has been associated with severe influenza A virus (IAV) infection. Here, we use an inhibitor of Toll-like receptor (TLR)3, a single-stranded oligonucleotide (ssON) with the capacity to inhibit certain endocytic routes, or a TLR3 agonist (synthetic double-stranded RNA PolyI:C), to evaluate modulation of innate responses during H1N1 IAV infection. Since IAV utilizes cellular endocytic machinery for viral entry, we also assessed ssON's capacity to affect IAV infection. We first show that IAV infected human monocyte-derived dendritic cells (MoDC) were unable to up-regulate the co-stimulatory molecules CD80 and CD86 required for T cell activation. Exogenous TLR3 stimulation did not overcome the IAV-mediated inhibition of co-stimulatory molecule expression in MoDC. However, TLR3 stimulation using PolyI:C led to an augmented pro-inflammatory cytokine response. We reveal that ssON effectively inhibited PolyI:C-mediated pro-inflammatory cytokine production in MoDC, notably, ssON treatment maintained an interferon response induced by IAV infection. Accordingly, RNAseq analyses revealed robust up-regulation of interferon-stimulated genes in IAV cultures treated with ssON. We next measured reduced IAV production in MoDC treated with ssON and found a length requirement for its anti-viral activity, which overlapped with its capacity to inhibit uptake of PolyI:C. Hence, in cases wherein an overreacting TLR3 activation contributes to IAV pathogenesis, ssON can reduce this signaling pathway. Furthermore, concomitant treatment with ssON and IAV infection in mice resulted in maintained weight and reduced viral load in the lungs. Therefore, extracellular ssON provides a mechanism for immune regulation of TLR3-mediated responses and suppression of IAV infection in vitro and in vivo in mice.

Protein association changes in the Hedgehog signaling complex mediate differential signaling strength.

Hedgehog (Hh) is a conserved morphogen that controls cell differentiation and tissue patterning in metazoans. In Drosophila, the Hh signal is transduced from the G protein-coupled receptor Smoothened (Smo) to the cytoplasmic Hh signaling complex (HSC). How activated Smo is translated into a graded activation of the downstream pathway is still not well understood. In this study, we show that the last amino acids of the cytoplasmic tail of Smo, in combination with G protein-coupled receptor kinase 2 (Gprk2), bind to the regulatory domain of Fused (Fu) and highly activate its kinase activity. We further show that this binding induces changes in the association of Fu protein with the HSC and increases the proximity of the Fu catalytic domain to its substrate, the Costal2 kinesin. We propose a new model in which, depending on the magnitude of Hh signaling, Smo and Gprk2 modulate protein association and conformational changes in the HSC, which are responsible for the differential activation of the pathway.

Single-Stranded Nucleic Acids Regulate TLR3/4/7 Activation through Interference with Clathrin-Mediated Endocytosis.

Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition, it was concentration dependent, and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream of TLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses.

Early Resistance of Non-virulent Mycobacterial Infection in C57BL/6 Mice Is Associated With Rapid Up-Regulation of Antimicrobial Cathelicidin Camp.

Early clearance of tuberculosis is the successful eradication of inhaled bacteria before the development of an adaptive immune response. We previously showed, by utilizing a non-virulent mycobacteria infection model, that C57BL/6 mice are more efficient than BALB/c in their control of bacterial growth in the lungs during the first weeks of the infection. Here, we assessed early (within 1-3 days) innate immune events locally in the lungs to identify factors that may contribute to the control of non-virulent mycobacterial burden. We confirmed that C57BL/6 mice are more resistant to infection compared with BALB/c after intranasal inoculation with mycobacterium. Transcriptomic analyses revealed a remarkably silent signature in C57BL/6 mice despite effective control of bacterial growth. In contrast, BALB/c mice up-regulated genes associated with neutrophil and myeloid cell chemotaxis and migration. Flow cytometry analyses corroborated the transcriptomic analyses and demonstrated influx of both neutrophil and myeloid cell populations in BALB/c mice, while these did not increase in C57BL/6 mice. We further detected increased release of TNF-α from BALB/c lung cells but limited release from C57BL/6-derived cells. However, C57BL/6 mice showed a marked early up-regulation of the Camp gene, encoding the cathelicidin CRAMP peptide, post-mycobacterial exposure. CRAMP (LL-37 in human) expression in the lungs was confirmed using immunofluorescence staining. Altogether, these findings show that C57BL/6 mice can clear the mycobacterial infection early and that this early control is associated with high CRAMP expression in the lungs without concomitant influx of immune cells. The role of CRAMP/LL-37 during mycobacterial infection may be relevant for novel protective strategies, and warrants further studies of human cohorts.

Dissolving microneedle delivery of nanoparticle-encapsulated antigen elicits efficient cross-priming and Th1 immune responses by murine Langerhans cells.

Dendritic cells (DCs) of the skin have an important role in skin-mediated immunity capable of promoting potent immune responses. We availed of polymeric dissolving microneedle (MN) arrays laden with nano-encapsulated antigen to specifically target skin DC networks. This modality of immunization represents an economic, efficient, and potent means of antigen delivery directly to skin DCs, which are inefficiently targeted by more conventional immunization routes. Following MN immunization, Langerhans cells (LCs) constituted the major skin DC subset capable of cross-priming antigen-specific CD8+ T cells ex vivo. Although all DC subsets were equally efficient in priming CD4+ T cells, LCs were largely responsible for orchestrating the differentiation of CD4+ IFN-γ- and IL-17-producing effectors. Importantly, depletion of LCs prior to immunization had a profound effect on CD8+ CTL responses in vivo, and vaccinated animals displayed reduced protective anti-tumor and viral immunity. Interestingly, this cross-priming bias was lost following MN immunization with soluble antigen, suggesting that processing and cross-presentation of nano-particulate antigen is favored by LCs. Therefore, these studies highlight the importance of LCs in skin immunization strategies and that targeting of nano-particulate immunogens through dissolvable polymeric MNs potentially provides a promising technological platform for improved vaccination strategies.

Macrophage- and neutrophil-derived TNF-α instructs skin langerhans cells to prime antiviral immune responses.

Dendritic cells are major APCs that can efficiently prime immune responses. However, the roles of skin-resident Langerhans cells (LCs) in eliciting immune responses have not been fully understood. In this study, we demonstrate for the first time, to our knowledge, that LCs in cynomolgus macaque skin are capable of inducing antiviral-specific immune responses in vivo. Targeting HIV-Gag or influenza hemagglutinin Ags to skin LCs using recombinant fusion proteins of anti-Langerin Ab and Ags resulted in the induction of the viral Ag-specific responses. We further demonstrated that such Ag-specific immune responses elicited by skin LCs were greatly enhanced by TLR ligands, polyriboinosinic polyribocytidylic acid, and R848. These enhancements were not due to the direct actions of TLR ligands on LCs, but mainly dependent on TNF-α secreted from macrophages and neutrophils recruited to local tissues. Skin LC activation and migration out of the epidermis are associated with macrophage and neutrophil infiltration into the tissues. More importantly, blocking TNF-α abrogated the activation and migration of skin LCs. This study highlights that the cross-talk between innate immune cells in local tissues is an important component for the establishment of adaptive immunity. Understanding the importance of local immune networks will help us to design new and effective vaccines against microbial pathogens.

Skin dendritic cell targeting via microneedle arrays laden with antigen-encapsulated poly-D,L-lactide-co-glycolide nanoparticles induces efficient antitumor and antiviral immune responses.

The efficacious delivery of antigens to antigen-presenting cells (APCs), in particular, to dendritic cells (DCs), and their subsequent activation remains a significant challenge in the development of effective vaccines. This study highlights the potential of dissolving microneedle (MN) arrays laden with nanoencapsulated antigen to increase vaccine immunogenicity by targeting antigen specifically to contiguous DC networks within the skin. Following in situ uptake, skin-resident DCs were able to deliver antigen-encapsulated poly-d,l-lactide-co-glycolide (PGLA) nanoparticles to cutaneous draining lymph nodes where they subsequently induced significant expansion of antigen-specific T cells. Moreover, we show that antigen-encapsulated nanoparticle vaccination via microneedles generated robust antigen-specific cellular immune responses in mice. This approach provided complete protection in vivo against both the development of antigen-expressing B16 melanoma tumors and a murine model of para-influenza, through the activation of antigen-specific cytotoxic CD8(+) T cells that resulted in efficient clearance of tumors and virus, respectively. In addition, we show promising findings that nanoencapsulation facilitates antigen retention into skin layers and provides antigen stability in microneedles. Therefore, the use of biodegradable polymeric nanoparticles for selective targeting of antigen to skin DC subsets through dissolvable MNs provides a promising technology for improved vaccination efficacy, compliance, and coverage.