Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation.

Bacterial DNA contains immunostimulatory CpG motifs that trigger an innate immune response capable of promoting host survival following infectious challenge. Yet CpG-driven immune activation may also have deleterious consequences, ranging from autoimmune disease to death. We find that repetitive elements present at high frequency in mammalian telomeres, but rare in bacteria, down-regulate CpG-induced immune activation. Suppressive activity correlates with the ability of telomeric TTAGGG repeats to form G-tetrads. Colocalization of CpG DNA with Toll-like receptor 9 in endosomal vesicles is disrupted by these repetitive elements, although cellular binding and uptake remain unchanged. These findings are the first to establish that specific host-derived molecules can down-regulate the innate immune response elicited by a TLR ligand

Effect of suppressive DNA on CpG-induced immune activation.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate a strong innate immune response. This stimulation can be abrogated by either removing the CpG DNA or adding inhibitory/suppressive motifs. Suppression is dominant over stimulation and is specific for CpG-induced immune responses (having no effect on LPS- or Con A-induced activation). Individual cells noncompetitively internalize both stimulatory and suppressive ODN. Studies using ODN composed of both stimulatory and suppressive motifs indicate that sequence recognition proceeds in a 5'-->3' direction, and that a 5' motif can block recognition of immediately 3' sequences. These findings contribute to our understanding of the immunomodulatory activity of DNA-based products and the rules that govern immune recognition of stimulatory and suppressive motifs

Immunological analysis of a Lactococcus lactis-based DNA vaccine expressing HIV gp120

For reasons of efficiency Escherichia coli is used today as the microbial factory for production of plasmid DNA vaccines. To avoid hazardous antibiotic resistance genes and endotoxins from plasmid systems used nowadays, we have developed a system based on the food-grade Lactococcus lactis and a plasmid without antibiotic resistance genes. We compared the L. lactis system to a traditional one in E. coli using identical vaccine constructs encoding the gp120 of HIV-1. Transfection studies showed comparable gp120 expression levels using both vector systems. Intramuscular immunization of mice with L. lactis vectors developed comparable gp120 antibody titers as mice receiving E. coli vectors. In contrast, the induction of the cytolytic response was lower using the L. lactis vector. Inclusion of CpG motifs in the plasmids increased T-cell activation more when the E. coli rather than the L. lactis vector was used. This could be due to the different DNA content of the vector backbones. Interestingly, stimulation of splenocytes showed higher adjuvant effect of the L. lactis plasmid. The study suggests the developed L. lactis plasmid system as new alternative DNA vaccine system with improved safety features. The different immune inducing properties using similar gene expression units, but different vector backbones and production hosts give information of the adjuvant role of the silent plasmid backbone. The results also show that correlation between the in vitro adjuvanticity of plasmid DNA and its capacity to induce cellular and humoral immune responses in mice is not straight forward

Systemic Treatment with CpG-B after Sublethal Rickettsial Infection Induces Mouse Death through Indoleamine 2,3-Dioxygenase (IDO)

Due to its strong immune stimulatory effects through TLR9, CpG-containing oligodeoxynucleotides (CpG ODN) have been tested in multiple clinical trials as vaccine adjuvant for infectious diseases and cancer. However, immune suppression induced by systemic administration of CpGs has been reported recently. In this study, we evaluated the impact of CpGs in an acute rickettsiosis model. We found that systemic treatment with type B CpG (CpG-B), but not type A CpG (CpG-A), at 2 days after sublethal R. australis infection induced mouse death. Although wild-type (WT) B6 and IDO−/− mice showed similar survival rates with three different doses of R. australis infection, treatment with CpG-B after sublethal infection consistently induced higher mortality with greater tissue bacterial loads in WT but not IDO−/− mice. Also, CpG-B treatment promoted the development of higher serum concentrations of proinflammatory cytokines/chemokines through IDO. Furthermore, while T cell-mediated immune responses enhanced by CpG-B were independent of IDO, treatment with CpG-B promoted T cell activation, PD-1 expression and cell apoptosis partially through IDO. A depletion study using anti-mPDCA-1 mAb indicated that plasmacytoid dendritic cells (pDC) were not required for CpG-B-induced death of R. australis-infected mice. Additionally, the results in iNOS−/− mice suggested that nitric oxide (NO) was partially involved in CpG-B-induced death of R. australis-infected mice. Surprisingly, pre-treatment with CpG-B before administration of a lethal dose of R. australis provided effective immunity in WT, IDO−/− and iNOS−/− mice. Taken together, our study provides evidence that CpGs exert complex immunological effects by both IDO-dependent and -independent mechanisms, and that systemic treatment with CpGs before or after infection has a significant and distinct impact on disease outcomes

CpG Immunotherapy in Chenopodium album sensitized mice: The comparison of IFN-gamma, IL-10 and IgE responses in intranasal and subcutaneous administrations

<p>Abstract</p> <p>Background</p> <p>Mucosal-based immunotherapy has been already used as an alternative form of allergen delivery. In asthma, the poor success rate of immune modulation could be a consequence of inadequate immune modulation in the airways. Previously, we have found that subcutaneous (S.C) co-administration of a homemade allergenic extract from Chenopodium album (Ch.a) pollen and Guanine-Cytosine containing deoxynucleotides (CpG-ODNs) is effective to prevent the inflammatory responses in mouse. In this study we used CpG/Ch.a for immunotherapy of Ch.a-induced asthma and compared the intranasal (I.N) and S.C routes of administration concerning IFN-γ, IL-10 and total IgE responses.</p> <p>Methods</p> <p>Ch.a sensitized mice were treated intranasaly or subcutaneously using CpG and Ch.a. extract. IFN-γ, IL-10 and total IgE were measured in supernatant culture of splenocytes and bronchoalveolor lavage (BAL) fluids by ELISA. Student's t test was used in the analysis of the results obtained from the test and control mice.</p> <p>Results</p> <p>We found that I.N administration of CpG/Ch.a in sensitized mice significantly increased the production of systemic and mucosal IFN-γ and IL-10 compared to phosphate buffered saline (PBS), Ch.a alone and control ODNs treated sensitized mice (P ≤ 0.001). On the other hand, S.C. route induced the systemic and mucosal IFN-γ in the lower levels than in I.N one, and failed to increase systemic IL-10 induction (P = 0.06). Total serum IgE in CpG/Ch.a treated mice in both routes showed significant decreases compared to three control groups (P ≤ 0.01). The amounts of IgE in BAL fluids were not measurable in all groups.</p> <p>Conclusion</p> <p>According to the results of this experiment we concluded that immunotherapy via the I.N co-administration of CpG/Ch.a in comparison with S.C route is more effective to stimulate the mucosal and regulatory responses in Ch.a induced asthma.</p

Antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine

Liposomes are versatile (sub)micron-sized membrane vesicles that can be used for a variety of applications, including drug delivery and in vivo imaging but they also represent excellent models for artificial membranes or cells. Several studies have demonstrated that in vitro transcription and translation can take place inside liposomes to obtain compartmentalized production of functional proteins within the liposomes (Kita et al. in Chembiochem 9(15):2403–2410, 2008; Moritani et al.in FEBS J, 2010; Kuruma et al. in Methods Mol Biol 607:161–171, 2010; Murtas et al. in Biochem Biophys Res Commun 363(1):12–17, 2007; Sunami et al. in Anal Biochem 357(1):128–136, 2006; Ishikawa et al. in FEBS Lett 576(3):387–390, 2004; Oberholzer et al. in Biochem Biophys Res Commun 261(2):238–241, 1999). Such a minimal artificial cell-based model is ideal for synthetic biology based applications. In this study, we propose the use of liposomes as artificial microbes for vaccination. These artificial microbes can be genetically programmed to produce specific antigens at will. To show proof-of-concept for this artificial cell-based platform, a bacterial in vitro transcription and translation system together with a gene construct encoding the model antigen β-galactosidase were entrapped inside multilamellar liposomes. Vaccination studies in mice showed that such antigen-expressing immunostimulatory liposomes (AnExILs) elicited higher specific humoral immune responses against the produced antigen (β-galactosidase) than control vaccines (i.e. AnExILs without genetic input, liposomal β-galactosidase or pDNA encoding β-galactosidase). In conclusion, AnExILs present a new platform for DNA-based vaccines which combines antigen production, adjuvanticity and delivery in one system and which offer several advantages over existing vaccine formulations

Patterns of Evolution and Host Gene Mimicry in Influenza and Other RNA Viruses

It is well known that the dinucleotide CpG is under-represented in the genomic DNA of many vertebrates. This is commonly thought to be due to the methylation of cytosine residues in this dinucleotide and the corresponding high rate of deamination of 5-methycytosine, which lowers the frequency of this dinucleotide in DNA. Surprisingly, many single-stranded RNA viruses that replicate in these vertebrate hosts also have a very low presence of CpG dinucleotides in their genomes. Viruses are obligate intracellular parasites and the evolution of a virus is inexorably linked to the nature and fate of its host. One therefore expects that virus and host genomes should have common features. In this work, we compare evolutionary patterns in the genomes of ssRNA viruses and their hosts. In particular, we have analyzed dinucleotide patterns and found that the same patterns are pervasively over- or under-represented in many RNA viruses and their hosts suggesting that many RNA viruses evolve by mimicking some of the features of their host's genes (DNA) and likely also their corresponding mRNAs. When a virus crosses a species barrier into a different host, the pressure to replicate, survive and adapt, leaves a footprint in dinucleotide frequencies. For instance, since human genes seem to be under higher pressure to eliminate CpG dinucleotide motifs than avian genes, this pressure might be reflected in the genomes of human viruses (DNA and RNA viruses) when compared to those of the same viruses replicating in avian hosts. To test this idea we have analyzed the evolution of the influenza virus since 1918. We find that the influenza A virus, which originated from an avian reservoir and has been replicating in humans over many generations, evolves in a direction strongly selected to reduce the frequency of CpG dinucleotides in its genome. Consistent with this observation, we find that the influenza B virus, which has spent much more time in the human population, has adapted to its human host and exhibits an extremely low CpG dinucleotide content. We believe that these observations directly show that the evolution of RNA viral genomes can be shaped by pressures observed in the host genome. As a possible explanation, we suggest that the strong selection pressures acting on these RNA viruses are most likely related to the innate immune response and to nucleotide motifs in the host DNA and RNAs

C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis with an annual incidence of approximately 2 million cases and is endemic in 88 countries, including Iran. CL's continued spread, along with rather ineffectual treatments and drug-resistant variants emergence has increased the need for advanced preventive strategies. We studied Type II cysteine proteinase (CPA) and Type I (CPB) with its C-terminal extension (CTE) as cocktail DNA vaccine against murine and canine leishmaniasis. However, adjuvants' success in enhancing immune responses to selected antigens led us to refocus our vaccine development programs. Herein, we discuss cationic solid lipid nanoparticles' (cSLN) ability to improve vaccine-induced protective efficacy against CL and subsequent lesion size and parasite load reduction in BALB/c mice. For this work, we evaluated five different conventional as well as novel parasite detection techniques, i.e., footpad imaging, footpad flowcytometry and lymph node flowcytometry for disease progression assessments. Vaccination with cSLN-cpa/cpb-CTE formulation showed highest parasite inhibition at 3-month post vaccination. Immunized mice showed reduced IL-5 level and significant IFN-ã increase, compared to control groups. We think our study represents a potential future and a major step forward in vaccine development against leishmaniasis

Cationic Liposomes Formulated with Synthetic Mycobacterial Cordfactor (CAF01): A Versatile Adjuvant for Vaccines with Different Immunological Requirements

It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed. (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge.CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection