New chimeric TLR7/NOD2 agonist is a potent adjuvant to induce mucosal immune responses.

BACKGROUND: PRR (Pattern Recognition Receptor) agonists have been widely tested as potent vaccine adjuvants. TLR7 (Toll-Like Receptor 7) and NOD2 (nucleotide-binding oligomerization domain 2) are key innate receptors widely expressed at mucosal levels. METHODS: Here, we evaluated the immunostimulatory properties of a novel hybrid chemical compound designed to stimulate both TLR7 and NOD2 receptors. FINDING: The combined TLR7/NOD2 agonist showed increase efficacy than TLR7L or NOD2L agonists alone or combined in different in vitro models. Dual TLR7/NOD2 agonist efficiently stimulates TLR7 and NOD2, and promotes the maturation and reprogramming of human dendritic cells, as well as the secretion of pro-inflammatory or adaptive cytokines. This molecule also strongly induces autophagy in human cells which is a major intracellular degradation system that delivers cytoplasmic constituents to lysosomes in both MHC class I and II-restricted antigen presentation. In vivo, TLR7/NOD2L agonist is a potent adjuvant after intranasal administration with NP-p24 HIV vaccine, inducing high-quality humoral and adaptive responses both in systemic and mucosal compartments. Use of TLR7/NOD2L adjuvant improves very significantly the protection of mice against an intranasal challenge with a vaccinia virus expressing the p24. INTERPRETATION: Dual TLR7/NOD2L agonist is a very potent and versatile vaccine adjuvant and promote very efficiently both systemic and mucosal immunity. FUNDING: This work was supported by Sidaction.

The STING ligand cGAMP potentiates the efficacy of vaccine-induced CD8+ T cells.

Pathogen recognition receptor (PRR) agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. Using an original in vitro approach to prime naive precursors from unfractionated human peripheral blood mononuclear cells, we assessed the influence of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a ligand for the stimulator of interferon genes (STING), on the induction of antigen-specific CD8+ T cells. We found that 2'3'-cGAMP and 3'3'-cGAMP were especially potent adjuvants in this system, driving the expansion and maturation of functionally replete antigen-specific CD8+ T cells via the induction of type I IFNs. The biological relevance of these findings was confirmed in vivo using two mouse models, in which 2'3'-cGAMP-adjuvanted vaccination elicited protective antitumor or antiviral CD8+ T cell responses. These results identify particular isoforms of cGAMP as effective adjuvants that may find utility in the development of novel immunotherapies and vaccines.

Cutting Edge: A Dual TLR2 and TLR7 Ligand Induces Highly Potent Humoral and Cell-Mediated Immune Responses.

TLR agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. The aim of this study was to evaluate the immunostimulatory properties of a novel compound incorporating covalently linked moieties designed to stimulate both TLR2 and TLR7. This dual TLR2/TLR7 agonist induced the maturation of dendritic cells and primed substantial populations of cytolytic and highly polyfunctional effector CD8+ T cells in vitro, and safely potentiated the immunogenic properties of a nanoparticulate Ag in vivo, eliciting humoral responses with a balanced TH1/TH2 profile in mice. Collectively, these data reveal the potential utility of chimeric adjuvants with synergistic activities mediated via TLRs.

Poly(Lactic Acid) Nanoparticles Targeting α5ß1 Integrin as Vaccine Delivery Vehicle, a Prospective Study.

Biodegradable polymeric nanoparticles are vehicles of choice for drug delivery and have the ability to encapsulate and present at their surface different molecules of interest. Among these bio-nanocarriers, poly(lactic acid) (PLA) nanoparticles have been used as adjuvant and vehicle for enhanced vaccine efficacy. In order to develop an approach to efficient vaccine delivery, we developed nanoparticles to target α5ß1 positive cells. We first overproduced, in bacteria, human fibronectin FNIII9/10 recombinant proteins possessing an integrin α5ß1 binding site, the RGDS sequence, or a mutated form of this site. After having confirmed the integrin binding properties of these recombinant proteins in cell culture assays, we were able to formulate PLA nanoparticles with these FNIII9/10 proteins at their surface. We then confirmed, by fluorescence and confocal microscopy, an enhanced cellular uptake by α5ß1+ cells of RGDS-FNIII9/10 coated PLA nanoparticles, in comparison to KGES-FNIII9/10 coated or non-coated controls. As a first vaccination approach, we prepared PLA nanoparticles co-coated with p24 (an HIV antigen), and RGDS- or KGES-FNIII9/10 proteins, followed by subcutaneous vaccine administration, in mice. Although we did not detect improvements in the apparent humoral response to p24 antigen in the serum of RGDS/p24 nanoparticle-treated mice, the presence of the FNIII proteins increased significantly the avidity index of anti-p24 antibodies compared to p24-nanoparticle-injected control mice. Future developments of this innovative targeted vaccine are discussed.

Biodegradable Polymeric Nanoparticles-Based Vaccine Adjuvants for Lymph Nodes Targeting.

Vaccines have successfully eradicated a large number of diseases. However, some infectious diseases (such as HIV, Chlamydia trachomatis or Bacillus anthracis) keep spreading since there is no vaccine to prevent them. One way to overcome this issue is the development of new adjuvant formulations which are able to induce the appropriate immune response without sacrificing safety. Lymph nodes are the site of lymphocyte priming by antigen-presenting cells and subsequent adaptive immune response, and are a promising target for vaccine formulations. In this review, we describe the properties of different polymer-based (e.g., poly lactic-co-glycolic acid, poly lactic acid …) particulate adjuvants as innovative systems, capable of co-delivering immunopotentiators and antigens. We point out how these nanoparticles enhance the delivery of antigens, and how their physicochemical properties modify their uptake by antigen-presenting cells and their migration into lymph nodes. We describe why polymeric nanoparticles increase the persistence into lymph nodes and promote a mature immune response. We also emphasize how nanodelivery directs the response to a specific antigen and allows the induction of a cytotoxic immune response, essential for the fight against intracellular pathogens or cancer. Finally, we highlight the interest of the association between polymer-based vaccines and immunopotentiators, which can potentiate the effect of the molecule by directing it to the appropriate compartment and reducing its toxicity.

Triggering Intracellular Receptors for Vaccine Adjuvantation.

Immune adjuvants are components that stimulate, potentiate, or modulate the immune response to an antigen. They are key elements of vaccines in both the prophylactic and therapeutic domains. In the past decade substantial progress in our understanding of innate immunity has paved the way for the design of next-generation adjuvants that stimulate a wide range of receptors. Within the framework of vaccine adjuvant design, this review outlines the interest of targeting endosomal and intracellular receptors to enhance and guide the immune response. We present and compare the molecules as well as potential combinations which are currently in the spotlight. We emphasize how targeting the appropriate receptor can direct immunity towards the appropriate response, such as a cytotoxic or mucosal response.

Cutting edge: New chimeric NOD2/TLR2 adjuvant drastically increases vaccine immunogenicity.

TLR ligands are critical activators of innate immunity and are being developed as vaccine adjuvants. However, their usefulness in conjunction with NOD-like receptor agonists remains poorly studied. In this study, we evaluated a new ligand that targets both TLR2 and NOD2 receptors. We assessed its ability to enhance dendritic cell maturation in vitro in addition to improving systemic and mucosal immune responses in mice. The chimeric NOD2/TLR2 ligand induced synergistic upregulation of dendritic cell maturation markers, costimulatory molecules, and secretion of proinflammatory cytokines compared with combinations of separate ligands. Furthermore, when coadministered with biodegradable nanoparticles carrying a model Ag, the ligand was able to induce high Ag-specific IgA and IgG titers at both systemic and mucosal sites after parenteral immunizations. These findings point out the potential utility of chimeric molecules TLR/NOD as adjuvants for vaccines to induce systemic and mucosal immune responses.

Engineering nicking enzymes that preferentially nick 5-methylcytosine-modified DNA.

N.ϕGamma is a strand-specific and site-specific DNA nicking enzyme (YCG↓GT or AC↑CGR). Here we describe the isolation of single and double mutants of N.ϕGamma with attenuated activity. The nicking domains (NDs) of E59A and 11 double mutants were fused to the 5mCG-binding domain of MBD2 and generated fusion enzymes that preferentially nick 5mCG-modified DNA. The CG dinucleotide can be modified by C5 methyltransferases (MTases) such as M.SssI, M.HhaI or M.HpaII to create composite sites AC↑YGG N(8-15) 5mCG. We also constructed a fusion enzyme 2xMBD2-ND(N.BceSVIII) targeting more frequent composite sites AS↑YS N(5-12) 5mCG in Mn2+ buffer. 5mCG-dependent nicking requires special digestion conditions in high salt (0.3 M KCl) or in Ni2+ buffer. The fusion enzyme can be used to nick and label 5mCG-modified plasmid and genomic DNAs with fluorescently labeled Cy3-dUTP and potentially be useful for diagnostic applications, DNA sequencing and optical mapping of epigenetic markers. The importance of the predicted catalytic residues D89, H90, N106 and H115 in N.ϕGamma was confirmed by mutagenesis. We found that the wild-type enzyme N.ϕGamma prefers to nick 5mCG-modified DNA in Ni2+ buffer even though the nicking activity is sub-optimal compared to the activity in Mg2+ buffer.

Structure determination and biochemical characterization of a putative HNH endonuclease from Geobacter metallireducens GS-15.

The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel ß-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15-20% amino acid sequence identity. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn²âº-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet_0936 may be a DNA repair enzyme.