mRNA-LNP COVID-19 Vaccine Lipids Induce Complement Activation and Production of Proinflammatory Cytokines: Mechanisms, Effects of Complement Inhibitors, and Relevance to Adverse Reactions.

A small fraction of people vaccinated with mRNA-lipid nanoparticle (mRNA-LNP)-based COVID-19 vaccines display acute or subacute inflammatory symptoms whose mechanism has not been clarified to date. To better understand the molecular mechanism of these adverse events (AEs), here, we analyzed in vitro the vaccine-induced induction and interrelations of the following two major inflammatory processes: complement (C) activation and release of proinflammatory cytokines. Incubation of Pfizer-BioNTech's Comirnaty and Moderna's Spikevax with 75% human serum led to significant increases in C5a, sC5b-9, and Bb but not C4d, indicating C activation mainly via the alternative pathway. Control PEGylated liposomes (Doxebo) also induced C activation, but, on a weight basis, it was ~5 times less effective than that of Comirnaty. Viral or synthetic naked mRNAs had no C-activating effects. In peripheral blood mononuclear cell (PBMC) cultures supplemented with 20% autologous serum, besides C activation, Comirnaty induced the secretion of proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1ß < TNF-α < IL-6 < IL-8. Heat-inactivation of C in serum prevented a rise in IL-1α, IL-1ß, and TNF-α, suggesting C-dependence of these cytokines' induction, although the C5 blocker Soliris and C1 inhibitor Berinert, which effectively inhibited C activation in both systems, did not suppress the release of any cytokines. These findings suggest that the inflammatory AEs of mRNA-LNP vaccines are due, at least in part, to stimulation of both arms of the innate immune system, whereupon C activation may be causally involved in the induction of some, but not all, inflammatory cytokines. Thus, the pharmacological attenuation of inflammatory AEs may not be achieved via monotherapy with the tested C inhibitors; efficacy may require combination therapy with different C inhibitors and/or other anti-inflammatory agents.

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue.

Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.

mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells.

mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables efficient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-of-experiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarifies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNA-LNP-based antitumor immunotherapy.

Lipid-Polyglutamate Nanoparticle Vaccine Platform.

Peptide-based subunit vaccines are attractive in view of personalized cancer vaccination with neo-antigens, as well as for the design of the newest generation of vaccines against infectious diseases. Key to mounting robust antigen-specific immunity is delivery of antigen to antigen-presenting (innate immune) cells in lymphoid tissue with concomitant innate immune activation to promote antigen presentation to T cells and to shape the amplitude and nature of the immune response. Nanoparticles that co-deliver both peptide antigen and molecular adjuvants are well suited for this task. However, in the context of peptide-based antigen, an unmet need exists for a generic strategy that allows for co-encapsulation of peptide and molecular adjuvants due to the stark variation in physicochemical properties based on the amino acid sequence of the peptide. These properties also strongly differ from those of many molecular adjuvants. Here, we devise a lipid nanoparticle (LNP) platform that addresses these issues. Key in our concept is poly(l-glutamic acid) (PGA), which serves as a hydrophilic backbone for conjugation of, respectively, peptide antigen (Ag) and an imidazoquinoline (IMDQ) TLR7/8 agonist as a molecular adjuvant. Making use of the PGA's polyanionic nature, we condensate PGA-Ag and PGA-IMDQ into LNP by electrostatic interaction with an ionizable lipid. We show in vitro and in vivo in mouse models that LNP encapsulation favors uptake by innate immune cells in lymphoid tissue and promotes the induction of Ag-specific T cells responses both after subcutaneous and intravenous administration.

The Opposing Effect of Type I IFN on the T Cell Response by Non-modified mRNA-Lipoplex Vaccines Is Determined by the Route of Administration.

mRNA-lipoplex vaccines are currently being explored in phase II clinical trials for the treatment of patients with advanced solid tumors. Mechanistically, these mRNA-lipoplex vaccines are characterized by the induction of type I interferon (IFN) centered innate responses. Earlier studies have identified type I IFNs as major regulators of the T cell response instigated by mRNA-lipoplex vaccines. However, stimulatory or, in contrast, profound inhibitory effects of type I IFNs were described depending on the study. In this mouse study, we demonstrated that the opposing roles of type I IFN signaling on the magnitude of the vaccine-evoked T cell responses is dependent on the route of mRNA-lipoplex administration and is regulated at the level of the T cells rather than indirectly through modulation of dendritic cell function. This study helps to understand the double-edged sword character of type I IFN induction upon mRNA-based vaccine treatment and may contribute to a more rational design of mRNA vaccination regimens.

Engineering Strategies for Lymph Node Targeted Immune Activation.

Development of vaccine technology that induces long lasting and potent adaptive immune responses is of vital importance to combat emerging pathogens and to design the next generation of cancer immunotherapies. Advanced biomaterials such as nanoparticle carriers are intensively explored to increase the efficacy and safety of vaccines and immunotherapies, based on their intrinsic potential to focus the therapeutic payload onto the relevant immune cells and to limit systemic distribution. With adaptive immune responses being primarily initiated in lymph nodes, the potency of nanoparticle vaccines in turn is tightly linked to their capacity to reach and accumulate in the lymph nodes draining the immunization site. Here, we discuss the main strategies applied to increase nanoparticle delivery to lymph nodes: (1) direct lymph node injection, (2) active cell-mediated transport through targeting of peripheral dendritic cells, and (3) exploiting passive transport through the afferent lymphatics.The intralymph nodal injection is obviously the most direct way for nanoparticles to reach lymph nodes, and multiple studies have demonstrated its capability in enhancing immunostimulant drugs' immune activation and increasing the therapeutic window. However, the requirement of using ultrasound guidance for mapping lymph nodes in patients renders intranodal administration unsuited for mass vaccination campaigns. As lymph nodes are fine structured organs with lymphocytes and chemokine gradients arrayed in a highly ordered fashion, the breakdown of such formats by the intralymph nodal injection is another concern. The exploitation of dendritic cells as live vectors for transporting nanoparticles to lymph nodes has intensively been studied both ex vivo and in vivo. While ex vivo engineering of dendritic cells in theory can achieve 100% dendritic cell-specific selectivity, a scenario impossible to be achieved in vivo, this procedure is usually laborious and complicated and entails the participation of professional staff and equipment. In addition, the poor efficiency of dendritic cell migration to the draining lymph node is another significant limitation following the injection of ex vivo cultured dendritic cells. Thus, in vivo targeting of surface receptors, particularly C-type lectin receptors, on dendritic cells by conjugating nanoparticles with antibodies or ligands is intensively studied by both academia and industry. Although such nanoparticles in vivo still face nonspecific engulfment by various phagocytes, multiple studies have shown its feasibility in targeting dendritic cells with high selectivity. Moreover, through optimizing the physicochemical properties of nanoparticles, nanoparticles can passively drain to lymph nodes carried by the interstitial flow. Compared to dendritic cell-mediated transport, passive draining is much faster and of higher efficiency. Of all such properties, size is the most important parameter as large particles (>500 nm) can only reach lymph nodes by an active cell-mediated transport. Other surface properties, such as the charge and the balance of hydrophobicity-vs-hydrophilicity, strongly influence the mobility of nanoparticles in the extracellular space. In addition, albumin, a natural fatty acid transporter, has recently been demonstrated capable of binding the amphiphiles through their lipid moiety and subsequent transporting them to lymph nodes.

mRNA Polyplexes with Post-Conjugated GALA Peptides Efficiently Target, Transfect, and Activate Antigen Presenting Cells.

Vaccines based on mRNA have emerged as potent systems to elicit CD8+ T cell responses against various cancers and viral infectious diseases. The efficient intracellular delivery of mRNA molecules encoding antigens into the cytosol of antigen-presenting cells (APCs) is still challenging, requiring cell attachment, active uptake, and subsequent endosomal escape. Here, we report a facile approach for the formulation of peptide-functionalized mRNA polyplexes using copper-free click chemistry to promote presentation of mRNA antigen by dendritic cells (DCs). After screening different membrane active peptides, GALA modified mRNA polyplexes (PPx-GALA) with a size around 350 nm and with a slightly negative surface charge (-7 mV), exhibited the highest EGFP-mRNA transfection in RAW 246.7 macrophages (∼36%) and D1 dendritic cells (∼50%) as compared to polyplexes decorated with melittin or LEDE peptides. Interestingly, we found that PPx-GALA enters DCs through sialic acid mediated endo/phagocytosis, which was not influenced by DC maturation. The PPx-GALA formulation exhibited 18-fold higher cellular uptake compared to a lipofectamine mRNA formulation without inducing cytotoxicity. Live cell imaging showed that PPx-GALA that were taken up by endocytosis induced calcein release from endosomes into the cytosol. DCs treated with PPx-GALA containing mRNA encoding for OVA displayed enhanced T cell responses and DC maturation. Collectively, these data provide a strong rationale for further study of this PPx-GALA formulation in vivo as a promising mRNA vaccine platform.

Modular core-shell polymeric nanoparticles mimicking viral structures for vaccination.

Recent advances in the development of protein-based vaccines have expanded the opportunities for preventing and treating both infectious diseases as well as cancer. However, the development of readily and efficient antigen delivery systems capable of stimulating strong cytotoxic T-lymphocyte (CTL) responses remains a challenge. With the attempt to closely mimic the properties of viruses in terms of their size and molecular organization, we constructed RNA (which is a ligand for Toll-like receptor 7 (TLR7) and TLR8) and antigen-loaded nanoparticles resembling the structural organization of viruses. Cationic polymers containing either azide or bicyclo[6.1.0]nonyne (BCN) groups were synthesized as electrostatic glue that binds negatively charged single stranded RNA (PolyU) to form a self-crosslinked polyplex core. An azide-modified model antigen (ovalbumin, OVA) and a BCN-modified mannosylated or galactosylated polymer were sequentially conjugated to the RNA core via disulfide bonds using copper free click chemistry to form the shell of the polyplexes. The generated reducible virus mimicking particles (VMPs) with a diameter of 200 nm and negatively surface charge (-14 mV) were colloidally stable in physiological conditions. The immunogenicity of these VMP vaccines was evaluated both in vitro and in vivo. The surface mannosylated VMPs (VMP-Man) showed 5 times higher cellular uptake by bone marrow derived DCs (BMDCs) compared to galactosylated VMP (VMP-Gal) counterpart. Moreover, VMP-Man efficiently activated DCs and greatly facilitated MHC I Ag presentation in vitro. Vaccination of mice with VMP-Man elicited strong OVA-specific CTL responses as well as humoral immune responses. These results demonstrate that the modular core-shell polymeric nanoparticles described in this paper are superior in inducing strong and durable immune responses compared to adjuvanted protein subunit vaccines and offer therefore a flexible platform for personalized vaccines.

Nanoparticle-Conjugate TLR7/8 Agonist Localized Immunotherapy Provokes Safe Antitumoral Responses.

Localized therapeutic modalities that subvert the tumor microenvironment from immune-suppressive to pro-immunogenic can elicit systemic antitumor immune responses that induce regression of directly treated as well as nontreated distal tumors. A key toward generating robust antitumor T cell responses is the activation of dendritic cells (DCs) in the tumor microenvironment. Treatment with agonists triggering various pattern recognition receptors is very efficient to activate DCs, yet suffers from the induction of serious immune-related adverse effects, which is closely linked to their unfavorable PK/PD profile causing systemic immune activation and cytokine release. Here, it is reported that nanoparticle conjugation of a highly potent TLR7/8 agonist restricts immune activation to the tumor bed and its sentinel lymph nodes without hampering therapeutic antitumor efficacy. On a mechanistic level, it is confirmed that localized treatment with a nanoparticle-conjugated TLR7/8 agonist leads to potent activation of DCs in the sentinel lymph nodes and promotes proliferation of tumor antigen-specific CD8 T cells. Furthermore, therapeutic improvement upon combination with anti-PDL1 checkpoint inhibition and Flt3L, a growth factor that expands and mobilizes DCs from the bone marrow, is demonstrated. The findings provide a rational base for localized tumor engineering by nanomedicine strategies that provide spatial control over immune-activation.

Lymph-Node-Targeted Immune Activation by Engineered Block Copolymer Amphiphiles-TLR7/8 Agonist Conjugates.

Small molecule immuno-modulators such as agonists of Toll-like receptors (TLRs) are attractive compounds to stimulate innate immune cells toward potent antiviral and antitumor responses. However, small molecules rapidly enter the systemic circulation and cause "wasted inflammation". Hence, synthetic strategies to confine their radius of action to lymphoid tissue are of great relevance, to both enhance their efficacy and concomitantly limit toxicity. Here, we demonstrate that covalent conjugation of a small molecule TLR7/8 agonist immunomodulatory to a micelle-forming amphiphilic block copolymer greatly alters the pharmacokinetic profile, resulting in highly efficient lymphatic delivery. Moreover, we designed amphiphilic block copolymers in such a way to form thermodynamically stable micelles through π-π stacking between aromatic moieties, and we engineered the block copolymers to undergo an irreversible amphiphilic to hydrophilic transition in response to the acidic endosomal pH.

Dendritic Cell Targeting mRNA Lipopolyplexes Combine Strong Antitumor T-Cell Immunity with Improved Inflammatory Safety.

In vitro transcribed mRNA constitutes a versatile platform to encode antigens and to evoke CD8 T-cell responses. Systemic delivery of mRNA packaged into cationic liposomes (lipoplexes) has proven particularly powerful in achieving effective antitumor immunity in animal models. Yet, T-cell responses to mRNA lipoplexes critically depend on the induction of type I interferons (IFN), potent pro-inflammatory cytokines, which inflict dose-limiting toxicities. Here, we explored an advanced hybrid lipid polymer shell mRNA nanoparticle (lipopolyplex) endowed with a trimannose sugar tree as an alternative delivery vehicle for systemic mRNA vaccination. Like mRNA lipoplexes, mRNA lipopolyplexes were extremely effective in conferring antitumor T-cell immunity upon systemic administration. Conversely to mRNA lipoplexes, mRNA lipopolyplexes did not rely on type I IFN for effective T-cell immunity. This differential mode of action of mRNA lipopolyplexes enabled the incorporation of N1 methyl pseudouridine nucleoside modified mRNA to reduce inflammatory responses without hampering T-cell immunity. This feature was attributed to mRNA lipopolyplexes, as the incorporation of thus modified mRNA into lipoplexes resulted in strongly weakened T-cell immunity. Taken together, we have identified lipopolyplexes containing N1 methyl pseudouridine nucleoside modified mRNA as potent yet low-inflammatory alternatives to the mRNA lipoplexes currently explored in early phase clinical trials.

Treatment with mRNA coding for the necroptosis mediator MLKL induces antitumor immunity directed against neo-epitopes.

Cancer immunotherapy can induce durable antitumor responses. However, many patients poorly respond to such therapies. Here we describe a generic antitumor therapy that is based on the intratumor delivery of mRNA that codes for the necroptosis executioner mixed lineage kinase domain-like (MLKL) protein. This intervention stalls primary tumor growth and protects against distal and disseminated tumor formation in syngeneic mouse melanoma and colon carcinoma models. Moreover, MLKL-mRNA treatment combined with immune checkpoint blockade further improves the antitumor activity. MLKL-mRNA treatment rapidly induces T cell responses directed against tumor neo-antigens and requires CD4+ and CD8+ T cells to prevent tumor growth. Type I interferon signaling and Batf3-dependent dendritic cells are essential for this mRNA treatment to elicit tumor antigen-specific T cell responses. Moreover, MLKL-mRNA treatment blunts the growth of human lymphoma in mice with a reconstituted human adaptive immune system. MLKL-based treatment can thus be exploited as an effective antitumor immunotherapy.

Post-PEGylated and crosslinked polymeric ssRNA nanocomplexes as adjuvants targeting lymph nodes with increased cytolytic T cell inducing properties.

Potent adjuvants are highly demanded for most protein and peptides based vaccine candidates in clinical development. Recognition of viral single stranded (ss)RNA by innate toll-like receptors 7/8 in dendritic cells results in a cytokine environment supportive to the establishment of long lasting antibody responses and Th1 oriented T cell immunity. To fully exploit the immunestimulatory properties of ssRNA, it needs to be adequately formulated to ensure its optimal delivery to dendritic cells in the vaccine draining lymph nodes. In the present paper, we report on the design of ssRNA nanocomplexes formed by complexation of the cationic poly(carbonic acid 2-dimethylamino-ethyl ester 1-methyl-2-(2-methacryloylamino)-ethyl ester) (pHPMA-DMAE) based polymeric carrier and ssRNA. The resulting ssRNA nanocomplexes were subsequently PEGylated through copper-free click chemistry using PEG-bicyclo[6.1.0]nonyne (PEG-BCN) and cross-linked via disulfide bonds to increase their stability. The obtained near-neutral charged PEGylated ssRNA nanocomplexes (~150 nm) combined ssRNA protection with highly efficient delivery of ssRNA to DCs in the vaccine draining lymph nodes after subcutanuously administration. When co-administrated with a model antigen (soluble ovalbumin (OVA)), ssRNA nanocomplexes were far more efficient at inducing CD8 cytolytic T cells when compared to OVA co-adminstarted with naked ssRNA. Furthermore, IgG2c antibody titers, indicative of Th1 skewed T cell responses, were >10 times increased by complexing ssRNA into the PEGylated nanocomplexes. This study highlights the potential of post-functionalizing ssRNA nanocomplexes by copper-free click chemistry and these findings indcate that this potent ssRNA adjuvant may profoundly improve the efficacy of a variety of vaccines requiring Th1-type immunity.

Potent anti-viral vaccine adjuvant based on pH-degradable nanogels with covalently linked small molecule imidazoquinoline TLR7/8 agonist.

Improving the immunogenicity of subunit vaccines, in particular skewing of the immune response towards Th1 type immunity, is crucial for the development of effective vaccines against intracellular infections and for the development of anti-cancer vaccines. Small molecule TLR7/8 agonist hold high potential for this purpose, but suffer from an undesirable pharmacokinetic profile, resulting in systemic inflammatory responses. An effective solution to this problem is covalent ligation to a larger carrier. Here, a degradable nanogel carrier containing a covalently linked imidazoquinoline (IMDQ) TLR7/8 agonist is explored as adjuvant for vaccination against the respiratory syncytial virus (RSV). In vitro and in vivo experiments in mice provide a solid rational base for preferring nanogels over soluble polymers as IMDQ carrier in terms of cellular uptake and lymph node accumulation.

Delivering Type I Interferon to Dendritic Cells Empowers Tumor Eradication and Immune Combination Treatments.

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects. Type I IFN has a long history in the treatment of cancer, but its multifaceted activity pattern and complex side effects prevent its clinical use. Here we develop AcTakines (Activity-on-Target cytokines), optimized (mutated) immunocytokines that are up to 1,000-fold more potent on target cells, allowing specific signaling in selected cell types only. Type I IFN-derived AcTaferon (AFN)-targeting Clec9A+ dendritic cells (DC) displayed strong antitumor activity in murine melanoma, breast carcinoma, and lymphoma models and against human lymphoma in humanized mice without any detectable toxic side effects. Combined with immune checkpoint blockade, chemotherapy, or low-dose TNF, complete tumor regression and long-lasting tumor immunity were observed, still without adverse effects. Our findings indicate that DC-targeted AFNs provide a novel class of highly efficient, safe, and broad-spectrum off-the-shelf cancer immunotherapeutics with no need for a tumor marker.Significance: Targeted type I interferon elicits powerful antitumor efficacy, similar to wild-type IFN, but without any toxic side effects. Cancer Res; 78(2); 463-74. ©2017 AACR.

Arginine-Rich Peptide-Based mRNA Nanocomplexes Efficiently Instigate Cytotoxic T Cell Immunity Dependent on the Amphipathic Organization of the Peptide.

To date, the mRNA delivery field has been heavily dominated by lipid-based systems. Reports on the use of nonlipid carriers for mRNA delivery in contrast are rare in the context of mRNA vaccination. This paper describes the potential of a cell-penetrating peptide containing the amphipathic RALA motif to deliver antigen-encoding mRNA to the immune system. RALA condenses mRNA into nanocomplexes that display acidic pH-dependent membrane disruptive properties. RALA mRNA nanocomplexes enable mRNA escape from endosomes and thereby allow expression of mRNA inside the dendritic cell cytosol. Strikingly, RALA mRNA nanocomplexes containing pseudouridine and 5-methylcytidine modified mRNA elicit potent cytolytic T cell responses against the antigenic mRNA cargo and show superior efficacy in doing so when compared to RALA mRNA nanocomplexes containing unmodified mRNA. RALA's unique sequence and structural organization are vital to act as mRNA vaccine vehicle, as arginine-rich peptide variants that lack the RALA motif show reduced mRNA complexation, impaired cellular uptake and lose the ability to transfect dendritic cells in vitro and to evoke T cell immunity in vivo.

Type I Interferons Modulate CD8+ T Cell Immunity to mRNA Vaccines.

mRNA vaccines have emerged as potent tools to elicit antitumor T cell immunity. They are characterized by a strong induction of type I interferons (IFNs), potent inflammatory cytokines affecting T cell differentiation and survival. Recent reports have attributed opposing roles for type I IFNs in modulating CD8+ T cell immunity to mRNA vaccines, from profoundly stimulatory to strongly inhibitory. The mechanisms behind this duality are unclear. Disentangling the factors governing the beneficial or detrimental impact of type I IFNs on CD8+ T cell responses is vital to the design of mRNA vaccines of increased potency. In light of recent advancements regarding the complex role of type I IFNs in regulating CD8+ T cell immunity to infectious diseases, we posit that the dual outcome of type I IFNs on CD8+ T cell responses to mRNA vaccination is determined by the timing and intensity of type I IFN induction relative to T cell receptor (TCR) activation.

Species-specific immunity to Helicobacter suis.

BACKGROUND: Helicobacter (H.) suis is mainly associated with pigs, but is also the most prevalent gastric non-H. pylori Helicobacter species found in humans. Both H. pylori and H. suis may cause persistent infection of the stomach. Several immune evasion mechanisms have been proposed for H. pylori, which focus to a great extent on its major virulence factors, which are absent in H. suis. The aim of this study was to gain more knowledge on immune evasion by H. suis. MATERIALS AND METHODS: Cytokine expression kinetics were monitored in the stomach of BALB/c mice experimentally infected with H. suis. The cytokine expression profile in the stomach of naturally H. suis-infected pigs was also determined. Subsequently, the effect of H. suis on murine and porcine dendritic cell (DC) maturation and their ability to elicit T-cell effector responses was analyzed. RESULTS: Despite a Th17/Th2 response in the murine stomach, the inflammatory cell influx was unable to clear H. suis infection. H. suis-stimulated murine bone marrow-derived dendritic cells induced IL-17 secretion by CD4+ cells in vitro. Natural H. suis infection in pigs evoked increased expression levels of IL-17 mRNA in the antrum and IL-10 mRNA in the fundus. In contrast to mice, H. suis-stimulated porcine monocyte-derived dendritic cells were unable to express MHCII molecules on their cell surface. These semimature DCs induced proliferation of T-cells, which showed an increased expression of TGF-ß and FoxP3 mRNA levels. CONCLUSIONS: Helicobacter suis might evade host immune responses by skewing toward a Treg-biased response.

Type I Interferons Interfere with the Capacity of mRNA Lipoplex Vaccines to Elicit Cytolytic T Cell Responses.

Given their high potential to evoke cytolytic T cell responses, tumor antigen-encoding messenger RNA (mRNA) vaccines are now being intensively explored as therapeutic cancer vaccines. mRNA vaccines clearly benefit from wrapping the mRNA into nano-sized carriers such as lipoplexes that protect the mRNA from degradation and increase its uptake by dendritic cells in vivo. Nevertheless, the early innate host factors that regulate the induction of cytolytic T cells to mRNA lipoplex vaccines have remained unresolved. Here, we demonstrate that mRNA lipoplexes induce a potent type I interferon (IFN) response upon subcutaneous, intradermal and intranodal injection. Regardless of the route of immunization applied, these type I IFNs interfered with the generation of potent cytolytic T cell responses. Most importantly, blocking type I IFN signaling at the site of immunization through the use of an IFNAR blocking antibody greatly enhanced the prophylactic and therapeutic antitumor efficacy of mRNA lipoplexes in the highly aggressive B16 melanoma model. As type I IFN induction appears to be inherent to the mRNA itself rather than to unique properties of the mRNA lipoplex formulation, preventing type I IFN induction and/or IFNAR signaling at the site of immunization might constitute a widely applicable strategy to improve the potency of mRNA vaccination.