Immunostimulatory CpG Motifs Research Articles

Immunostimulatory DNA Hydrogel Enhances Protective Efficacy of Nanotoxoids against Bacterial Infection.

While vaccines have been highly successful in protecting against various infections, there are still many high-priority pathogens for which there are no clinically approved formulations. To overcome this challenge, researchers have explored the use of nanoparticulate strategies for more effective antigen delivery to the immune system. Along these lines, nanotoxoids are a promising biomimetic platform that leverages cell membrane coating technology to safely deliver otherwise toxic bacterial antigens in their native form for antivirulence vaccination. Here, in order to further boost their immunogenicity, nanotoxoids formulated against staphylococcal α-hemolysin are embedded into a DNA-based hydrogel with immunostimulatory CpG motifs. The resulting nanoparticle-hydrogel composite is injectable and improves the in vivo delivery of vaccine antigens while simultaneously stimulating nearby immune cells. This leads to elevated antibody production and stronger antigen-specific cellular immune responses. In murine models of pneumonia and skin infection caused by methicillin-resistant Staphylococcus aureus, mice vaccinated with the hybrid vaccine formulation are well-protected. This work highlights the benefits of combining nanoparticulate antigen delivery systems with immunostimulatory hydrogels into a single platform, and the approach can be readily generalized to a wide range of infectious diseases.

Role of Interleukin-6 in the Antigen-Specific Mucosal Immunoglobulin A Responses Induced by CpG Oligodeoxynucleotide-Loaded Cationic Liposomes.

An advantage of mucosal vaccines over conventional parenteral vaccines is that they can induce protective immune responses not only at mucosal surfaces but also in systemic compartments. Despite this advantage, few live attenuated or inactivated mucosal vaccines have been developed and applied clinically. We recently showed that the intranasal immunization of ovalbumin (OVA) with class B synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motif (CpG ODN)-loaded cationic liposomes synergistically exerted both antigen-specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) responses in mice. However, the mechanism underlying the mucosal adjuvant activity of CpG ODN-loaded liposomes remains unknown. In the present study, we showed that the intranasal administration of CpG ODN-loaded cationic liposomes elicited interleukin (IL)-6 release in nasal tissues. Additionally, pre-treatment with an anti-IL-6 receptor (IL-6R) antibody attenuated antigen-specific nasal IgA production but not serum IgG responses. Furthermore, the intranasal administration of OVA and CpG ODN-loaded cationic liposomes increased the number of IgA+/CD138+ plasma cells and IgA+/B220+ B cells in the nasal passages. This increase was markedly suppressed by pre-treatment with anti-IL-6R blocking antibody. In conclusion, IL-6 released by CpG ODN-loaded cationic liposomes at the site of administration may play a role in the induction of antigen-specific IgA responses by promoting differentiation into IgA+ plasma cells for IgA secretion from B cells.

Baculovirus Vectors Induce the Production of Interferons in Swine: Their Potential in the Development of Antiviral Strategies.

The huge variety of viruses affecting swine represents a global threat. Since vaccines against highly contagious viruses last several days to induce protective immune responses, antiviral strategies for rapid control of outbreak situations are needed. The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), an insect virus, has been demonstrated to be an effective vaccine vector for mammals. Besides the ability to display or transduce heterologous antigens, it also induces strong innate immune responses and provides IFN-mediated protection against lethal challenges with viruses like foot-and-mouth disease virus (FMDV) in mice. Thus, the aim of this study was to evaluate the ability of AcMNPV to induce IFN production and elicit antiviral activity in porcine peripheral blood mononuclear cells (PBMCs). Our results demonstrated that AcMNPV induced an IFN-α-mediated antiviral activity in PBMCs in vitro. Moreover, the inoculation of AcMNPV in piglets led to the production of type I and II IFNs in sera from inoculated animals and antiviral activities against vesicular stomatitis virus (VSV) and FMDV measured by in vitro assays. Finally, it was demonstrated that the pseudotyping of AcMNPV with VSV-G protein, but not the enrichment of the AcMNPV genome with specific immunostimulatory CpG motifs for the porcine TLR9, improved the ability to induce IFN-α production in PBMCs in vitro. Together, these results suggest that AcMNPV is a promising tool for the induction of IFNs in antiviral strategies, with the potential to be biotechnologically improved.

Kiwifruit drives human microbiota-derived DNA to stimulate IL-7 secretion in intestinal epithelial cells

Abstract Intestinal immune tolerance is maintained through the interplay between commensal microbiota, immune cells and epithelium. Constitutive intestinal epithelial-secreted interleukin (IL)-7 facilitates the proliferation and thus maintenance of gamma-delta T lymphocytes, which preserves intestinal mucosal immune surveillance. Since IL-7 is essential for maintaining intestinal health, we hypothesised that microbiota-derived components influenced by dietary kiwifruit support the intestinal IL-7 expression. In vitro gastro-ileal digestion and colonic fermentation of gold and green kiwifruit generated soluble bacteria-free fermenta, notably bacterial DNA that modulated basal and interferon gamma-stimulated IL-7 secretion in HT-29 cells. Epithelial IL-7 secretion correlated positively with total bacterial DNA, and the DNA originated from bifidobacteria, which are known to contain immunostimulatory CpG motifs. These findings demonstrate the involvement of microbiota DNA in promoting IL-7 secretion in the intestinal epithelium and contribute to unravelling a mechanism by which kiwifruit serve to maintain intestinal immune homeostasis.

Abstract 4782: STACT: A novel tumor-targeting, systemically-administered delivery platform capable of targeting intractable pathways and precise immunomodulation of the tumor microenvironment

Abstract We have engineered a tumor-targeted, microbial immunotherapy platform called STACT (Salmonella Typhimurium (Attenuated) and Checkpoint Therapy). The platform strain has been engineered to increase tumor-specific enrichment, reduce immunosuppressive inflammation and enhance tolerability. Upon phagocytosis by tumor-resident immune cells, the microbe delivers plasmid DNA, which can either encode inhibitory microRNAs to knockdown immune targets of interest, or encode immuno-modulatory cDNA expression cassettes, alone or in specific combinations. The STACT platform strain is over 10,000-fold attenuated for virulence due to disruptions in the msbB and purI genes, that result in the detoxification of the surface LPS and purine/adenosine auxotrophy, respectively. The STACT platform strain required addition of exogenous adenosine or purines at concentrations found in the tumor micro-environment, but not in healthy tissue for replication in vitro. The STACT platform strain was unable to replicate significantly in infected macrophage cell lines, but was able to colonize tumors in mice and deliver functional plasmids. This strain was further engineered by precise genome deletions of the fljB and fliC genes, encoding the bacterial flagellar subunits that are strong TLR5 agonists and induce inflammasome-mediated pyroptosis in macrophages. Deletion of the flagellin genes prevented bacterial cell motility but did not affect tumor-specific colonization after IV administration, and tumor enrichment was observed at levels over 100,000 times greater than in spleens. A plasmid maintenance system was engineered by deletion of the chromosomal asd gene and complementation of the asd gene on a copy-number optimized plasmid to ensure plasmid maintenance in vivo. The asd system allowed for significantly improved plasmid retention in vivo without antibiotic selection. Furthermore, the vector incorporates immunostimulatory CpG motifs into the strain to help promote a TLR9-mediated adaptive immune response to tumor antigens. A library of inhibitory RNAi’s against a set of immuno-modulatory targets, including TREX1, PD-L1, and TGF-beta were screened for optimal knockdown of gene expression by qPCR and western blot. Pairwise combinatorial knockdown of specific targets was also observed in human cells. IV administration of STACT encoding TGF-beta RNAi demonstrated significant tumor growth inhibition in a subcutaneous tumor model. STACT is a highly attenuated microbial immunotherapy platform engineered to deliver immunomodulatory molecules to phagocytic cells in the tumor microenvironment after systemic administration. This platform can be engineered to knock down combinations of immune checkpoints or express immunostimulatory genes in a single therapeutic modality. Citation Format: Chris S. Rae, Marina Besprozvannaya, John Faulhaber, Anastasia M. Makarova, Beverly King, Laura Hix Glickman, Christopher D. Thanos, Justin Skoble. STACT: A novel tumor-targeting, systemically-administered delivery platform capable of targeting intractable pathways and precise immunomodulation of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4782.

Experimental Combined Immunotherapy of Tumours with Major Histocompatibility Complex Class I Downregulation.

Combined immunotherapy constitutes a novel, advanced strategy in cancer treatment. In this study, we investigated immunotherapy in the mouse TC-1/A9 model of human papillomavirus type 16 (HPV16)-associated tumors characterized by major histocompatibility complex class I (MHC-I) downregulation. We found that the induction of a significant anti-tumor response required a combination of DNA vaccination with the administration of an adjuvant, either the synthetic oligodeoxynucleotide ODN1826, carrying immunostimulatory CpG motifs, or α-galactosylceramide (α-GalCer). The most profound anti-tumor effect was achieved when these adjuvants were applied in a mix with a one-week delay relative to DNA immunization. Combined immunotherapy induced tumor infiltration with various subsets of immune cells contributing to tumor regression, of which cluster of differentiation (CD) 8+ T cells were the predominant subpopulation. In contrast, the numbers of tumor-associated macrophages (TAMs) were not markedly increased after immunotherapy but in vivo and in vitro results showed that they could be repolarized to an anti-tumor M1 phenotype. A blockade of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) immune checkpoint had a negligible effect on anti-tumor immunity and TAMs repolarization. Our results demonstrate a benefit of combined immunotherapy comprising the activation of both adaptive and innate immunity in the treatment of tumors with reduced MHC-I expression.

Linear doggybone DNA vaccine induces similar immunological responses to conventional plasmid DNA independently of immune recognition by TLR9 in a pre-clinical model

Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours.

Impact of viral and host DNA methylations on HPV16-related cervical cancer pathogenesis.

Epigenetic alterations within human papillomavirus (HPV) and host cellular genomes are known to occur during cervical carcinogenesis. Our objective was to analyse the influence of (1) methylation within two immunostimulatory CpG motifs within HPV16 E6 and E7 genes around the viral late promoter and their correlation, if any, with expression deregulation of host receptor (TLR9) and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and (2) global DNA methylation levels within CpGs of the repetitive Alu sequences, on cervical cancer (CaCx) pathogenesis. Significantly higher proportions of CaCx samples portrayed methylation in immunostimulatory CpG motifs, compared to HPV16-positive non-malignant samples, with cases harbouring episomal HPV16 showing decreased methylation compared to those with viral integration. A significant linear trend of TLR9 upregulation was recorded in the order of HPV-negative controls < HPV16-positive non-malignant samples < HPV16-positive CaCx cases. TLR9 upregulation in cases with episomal HPV16 was again higher among those with non-methylated immunostimulatory CpG motifs. Comparison of cases with HPV-negative controls revealed that DNMT3A was significantly downregulated only among integrated cases, DNMT3B was significantly overexpressed among both categories of cases, although at variable levels, while DNMT1 failed to show any deregulated expression among the cases. Global host DNA hypomethylation, also showed a significant linear increasing trend through the progressive CaCx development stages mentioned above and was most prominently higher among cases with episomal HPV16 as opposed to viral integration. Thus, HPV16 and host methylations appear to influence CaCx pathogenesis, with differential molecular signatures among CaCx cases with episomal and integrated HPV16.

Attachment of class B CpG ODN onto DOTAP/DC-chol liposome in nasal vaccine formulations augments antigen-specific immune responses in mice

BackgroundTo overcome infectious diseases, the development of mucosal vaccines would be an effective strategy, since mucosal surfaces are the entry site for most pathogens. In general, protein antigens show inherently poor immunogenicity when administered by the mucosal route. Therefore, co-administration of an appropriate mucosal adjuvant is required to exert immune responses toward pathogen-derived antigens effectively. However, the development of a safe and effective mucosal adjuvant system is still challenging. Although, recent studies reported that oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motifs (CpG ODNs) act as potent mucosal adjuvants and are useful in the formulation of nasal vaccines, there are some disadvantages. For instance, the administration of phosphorothioate (PS)-modified CpG ODNs can induce adverse systemic effects, such as splenomegaly, in a dose-dependent manner. Therefore, a reduced dose of CpG ODN might be crucial when used as vaccine adjuvant for clinical purposes. Therefore, we prepared a CpG ODN-loaded cationic liposome, and evaluated its mucosal adjuvant activity.ResultsWe prepared a CpG ODN-loaded DOTAP/DC-chol liposome that was stable during our experiments, by mixing CpG ODNs and liposomes at an N/P ratio of 4. Further, we demonstrated that the attachment of class B CpG ODN to the DOTAP/DC-chol liposomes synergistically enhanced antigen-specific IgA production in the nasal area than that induced by CpG ODN and DOTAP/DC-chol liposomes alone. The endpoint titers were more than tenfolds higher than that induced by either single CpG ODN or single DOTAP/DC-chol liposomes. Additionally, although serum IgG1 responses (indicated as a Th2 response) remained unchanged for DOTAP/DC-chol liposomes and CpG ODN-loaded DOTAP/DC-chol liposomes, the CpG ODN-loaded DOTAP/DC-chol liposomes synergistically induced the production of serum IgG2a (indicated as a Th1 response) than that by the individual liposomes.ConclusionsWe conclude that the advantage of using DOTAP/DC-chol liposome harboring CpG ODN is it induces both antigen-specific mucosal IgA responses and balanced Th1/Th2 responses. Therefore, such a combination enables us to resolve the adverse effects of using CpG ODNs (as a mucosal adjuvant) by reducing the overall dose of CpG ODNs. Further, the biodegradable and essentially non-antigenic nature of the liposomes makes it superior than the other existing mucosal adjuvants.

A Tetravalent Formulation Based on Recombinant Nucleocapsid-like Particles from Dengue Viruses Induces a Functional Immune Response in Mice and Monkeys.

Despite the considerable effort that has been invested in elucidating the mechanisms of protection and immunopathogenesis associated with dengue virus infections, a reliable correlate of protection against the disease remains to be found. Neutralizing Abs, long considered the prime component of a protective response, can exacerbate disease severity when present at subprotective levels, and a growing body of data is challenging the notion that their titers are positively correlated with disease protection. Consequently, the protective role of cell-mediated immunity in the control of dengue infections has begun to be studied. Although earlier research implicated cellular immunity in dengue immunopathogenesis, a wealth of newer data demonstrated that multifunctional CD8+ T cell responses are instrumental for avoiding the more severe manifestations of dengue disease. In this article, we describe a new tetravalent vaccine candidate based on recombinant dengue virus capsid proteins, efficiently produced in Escherichia coli and purified using a single ion-exchange chromatography step. After aggregation to form nucleocapsid-like particles upon incubation with an oligodeoxynucleotide containing immunostimulatory CpG motifs, these Ags induce, in mice and monkeys, an IFN-γ-secreting cell response that significantly reduces viral load after challenge without the contribution of antiviral Abs. Therefore, this new vaccine candidate may not carry the risk for disease enhancement associated with Ab-based formulations.

Nanodelivery systems for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Synthetic oligodeoxynucleotides containing immunostimulatory CpG motif mimic bacterial DNA and are potent activator of innate and adaptive immune responses. Therefore, CpG ODNs have significant potentials as immunotherapeutic agent for treatment of infectious diseases, allergy and cancer. Many clinical trials involving CpG ODNs either used alone or as adjuvant have been initiated. However, delivery of CpG ODNs to target sites still remains a great challenge due to their extreme susceptibility to nuclease degradation in serum and poor cellular uptake. Chemical modification of CpG ODNs backbone can protect them against degradation by nucleases, but have raised concern regarding several severe side effects. Development of efficient CpG ODNs delivery systems to address these issues and enhance their immunostimulatory effect are highly desirable. In recent years, the emergence of nanotechnology has provided unprecedented opportunities to encapsulate CpG ODN into various nanocarriers or synthesize CpG ODNs nanostructures. This review provides an overview of the delivery systems based on nanomaterials and nanostructures newly developed for enhancing the immunostimulatory effect of CpG ODNs, together with a brief discussion on perspectives for future studies in this field.

Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release.

Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis

Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article supports the hypothesis that in the absence of invasive proteins or relevant chemical treatment, L. lactis was found efficient in delivering DNA to mammalian cells.

Therapeutic Implications of Orally Delivered Immunomodulatory Oligonucleotides

DNA has multiple and complex effects on the immune system. Bacterial DNA contains immunostimulatory CpG motifs that trigger cells expressing Toll-like receptor 9 (TLR9) to proliferate, mature, and support T helper type 1 (Th1)-biased immune responses (reviewed in ref. 1). This response confers a selective advantage to the host by improving resistance to infection. In contrast, mammalian DNA contains sequence motifs that downregulate the immune system. Synthetic oligodeoxynucleotides that mimic this inhibitory activity (iODNs) slow or prevent the development of inflammatory and autoimmune diseases, consistent with the release of DNA from dying host cells inhibiting autoaggressive immune responses (reviewed in ref. 2). As reported in this issue, Wang et al. break new ground in the study of immunomodulatory ODNs by showing that the severity of chemically induced atopic dermatitis can be reduced by oral delivery of iODNs and worsened by CpG ODNs.3

Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines.

X-ray scattering from immunostimulatory tetrapod-shaped DNA in aqueous solution to explore its biological activity-conformation relationship.

We carried out synchrotron X-ray scattering experiments from four DNA supermolecules designed to form tetrapod shapes; these supermolecules had different sequences but identical numbers of total base pairs, and each contained an immunostimulatory CpG motif. We confirmed that the supermolecules did indeed form the expected tetrapod shape. The sample that had the largest radius of gyration (Rg) induced the most cytokine secretion from cultured immune cells. Structural analysis in combination with a rigid tetrapod model and an atomic scale DNA model revealed that the larger Rg can be ascribed to dissociation of the DNA double strands in the central connecting portion of the DNA tetrapod. This finding suggests that the biological activity is related to the ease with which single DNA strands can be formed.

Genomic CpG Enrichment of Oncolytic Parvoviruses as a Potent Anticancer Vaccination Strategy for the Treatment of Pancreatic Adenocarcinoma

Objective: For quite a long time oncolytic viruses (OVs) have been regarded merely as specific tumor cell killers, while disregarding the fact that all oncolytic activities take place in the context of a functional immune system. Oncolytic parvoviruses (PV) represent non-pathogenic, naturally oncolytic (non-modified), animal (rodent) viruses with a tropism that extends to a number of transformed human cells. Our recent work using various animal models substantiates the contention that H-1PV acts as both an oncolytic agent and an adjuvant, by direct cytoreduction in the tumor and bystander antitumor immunity. ImmunostimulatoryCpG motifs were incorporated into the singlestranded DNA genome of H-1PV and our current objective was to test whether the CpG-armed virus was in possession of an enhanced adjuvant capacity. Methods: The immunogenic potential of the CpG-enriched parvoviral derivative (JabCG) was tested in in vitro infection of human PBMCs or coculture of DCs and T-cells. In vivo tumor xenografts were raised in NOD.SCID mice that were later reconstituted with an autologous DCs and T-cells mix primed with an infected or chemovirotherapy (gemcitabine and H-1PV)-treated pancreatic cancer line vaccine. The therapeutic activity of the native and modified viruses was evaluated upon systemic application in pancreatic cancer-bearing immunocompetent Lewis rats. Results: Compared with wt H-1PV, JabCG displayed enhanced immunotherapeutic capacity to activate human immune cells ex vivo (PBMCs or DCs and T-cells isolated from pancreatic cancer patients) with a striking increase in the capacity of the latter cells for suppressing autologous tumorxenografts in NOD.SCID mice. Furthermore, intravenous application of JabCG in immunocompetent rats caused early NK and T-cell infiltration into tumors, elevated IFNγ levels in serum and spleens, and notably prolonged survival, as compared to control-treated animals. Conclusion: Taken together, data indicate that CpG-enrichment of OVs represents a potent strategy to enhance their immunotherapeutic properties.

Immunostimulatory CpG motifs in the genomes of gut bacteria and their role in human health and disease

Toll-like receptor (TLR) signalling plays an important role in epithelial and immune cells of the intestine. TLR9 recognizes unmethylated CpG motifs in bacterial DNA, and TLR9 signalling maintains the gut epithelial homeostasis. Here, we carried out a bioinformatic analysis of the frequency of CpG motifs in the genomes of gut commensal bacteria across major bacterial phyla. The frequency of potentially immunostimulatory CpG motifs (all CpG hexamers) or purine-purine-CG-pyrimidine-pyrimidine hexamers was linearly dependent on the genomic G+C content. We found that species belonging to Proteobacteria, Bacteroidetes and Actinobacteria (including bifidobacteria) carried high counts of GTCGTT, the optimal motif stimulating human TLR9. We also found that Enterococcus faecalis, Lactobacillus casei, Lactobacillus plantarum and Lactobacillus rhamnosus, whose strains have been marketed as probiotics, had high counts of GTCGTT motifs. As gut bacterial species differ significantly in their genomic content of CpG motifs, the overall load of CpG motifs in the intestine depends on the species assembly of microbiota and their cell numbers. The optimal CpG motif content of microbiota may depend on the host's physiological status and, consequently, on an adequate level of TLR9 signalling. We speculate that microbiota with increased numbers of microbes with CpG motif-rich DNA could better support mucosal functions in healthy individuals and improve the T-helper 1 (Th1)/Th2 imbalance in allergic diseases. In autoimmune disorders, CpG motif-rich DNA could, however, further increase the Th1-type immune responsiveness. Estimation of the load of microbe-associated molecular patterns, including CpG motifs, in gut microbiota could shed new light on host-microbe interactions across a range of diseases.

Targeted nanoparticle delivery overcomes off-target immunostimulatory effects of oligonucleotides and improves therapeutic efficacy in chronic lymphocytic leukemia

AbstractSeveral RNA-targeted therapeutics, including antisense oligonucleotides (ONs), small interfering RNAs, and miRNAs, constitute immunostimulatory CpG motifs as an integral part of their design. The limited success with free antisense ONs in hematologic malignancies in recent clinical trials has been attributed to the CpG motif–mediated, TLR-induced prosurvival effects and inefficient target modulation in desired cells. In an attempt to diminish their off-target prosurvival and proinflammatory effects and specific delivery, as a proof of principle, in the present study, we developed an Ab-targeted liposomal delivery strategy using a clinically relevant CD20 Ab (rituximab)–conjugated lipopolyplex nanoparticle (RIT-INP)– and Bcl-2–targeted antisense G3139 as archetypical antisense therapeutics. The adverse immunostimulatory responses were abrogated by selective B cell–targeted delivery and early endosomal compartmentalization of G3139-encapsulated RIT-INPs, resulting in reduced NF-κB activation, robust Bcl-2 down-regulation, and enhanced sensitivity to fludarabine-induced cytotoxicity. Furthermore, significant in vivo therapeutic efficacy was noted after RIT-INP–G3139 administration in a disseminated xenograft leukemia model. The results of the present study demonstrate that CD20-targeted delivery overcomes the immunostimulatory properties of CpG-containing ON therapeutics and improves efficient gene silencing and in vivo therapeutic efficacy for B-cell malignancies. The broader implications of similar approaches in overcoming immunostimulatory properties of RNA-directed therapeutics in hematologic malignancies are also discussed.

CpG ODN and ISCOMATRIX Adjuvant: A Synergistic Adjuvant Combination Inducing Strong T-Cell IFN-γResponses

For the induction of robust humoral and cellular immune responses, a strong rationale exists to use vaccine-adjuvant combinations possessing both immune modulatory and enhanced delivery capabilities. Herein, we evaluated the combination of 2 different adjuvants, a TLR9 agonist, composed of synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG), and ISCOMATRIX adjuvant (ISCOMATRIX), composed of saponin, phospholipid, and cholesterol, which possesses both immunostimulatory and delivery properties. While both individual adjuvants have been shown effective in numerous preclinical and clinical studies, it is likely that for optimal adjuvant activity a combined adjuvant approach will be necessary. Herein, using three different antigens, namely, hepatitis B surface antigen (HBsAg), ovalbumin (OVA), and influenza A haemagglutinin antigen (HA), we show in mice that some adjuvant effects of CpG and ISCOMATRIX are further enhanced if they are used in combination. In particular, with all three antigens, IFN-γ levels were greatly increased with the CpG/ISCOMATRIX combination. The ability of the CpG/ISCOMATRIX combination to induce antitumor responses when administered with OVA following administration to mice of a highly metastatic OVA-secreting tumor cell line (B16-OVA melanoma) was also demonstrated. Thus the CpG/ISCOMATRIX combination may prove to be a valuable tool in the development of novel or improved vaccines.