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• W2026083914 abstract "The use of DNA and viral vector-based vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to the lack of their genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study, we evaluated the potency of antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE) as a novel vaccination approach. We demonstrate that subcutaneous immunization of mice with mRNA encoding the HIV-1 antigen Gag complexed with DOTAP/DOPE elicits antigen-specific, functional T cell responses resulting in specific killing of Gag peptide-pulsed cells and the induction of humoral responses. In addition, we show that DOTAP/DOPE complexed antigen-encoding mRNA displays immune-activating properties characterized by secretion of type I interferon (IFN) and the recruitment of proinflammatory monocytes to the draining lymph nodes. Finally, we demonstrate that type I IFN inhibit the expression of DOTAP/DOPE complexed antigen-encoding mRNA and the subsequent induction of antigen-specific immune responses. These results are of high relevance as they will stimulate the design and development of improved mRNA-based vaccination approaches. The use of DNA and viral vector-based vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to the lack of their genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study, we evaluated the potency of antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE) as a novel vaccination approach. We demonstrate that subcutaneous immunization of mice with mRNA encoding the HIV-1 antigen Gag complexed with DOTAP/DOPE elicits antigen-specific, functional T cell responses resulting in specific killing of Gag peptide-pulsed cells and the induction of humoral responses. In addition, we show that DOTAP/DOPE complexed antigen-encoding mRNA displays immune-activating properties characterized by secretion of type I interferon (IFN) and the recruitment of proinflammatory monocytes to the draining lymph nodes. Finally, we demonstrate that type I IFN inhibit the expression of DOTAP/DOPE complexed antigen-encoding mRNA and the subsequent induction of antigen-specific immune responses. These results are of high relevance as they will stimulate the design and development of improved mRNA-based vaccination approaches. Over the past decades, nucleic acid-based vaccines have emerged as a promising approach for the effective induction of antigen-specific cellular immune responses. As concerns have been raised regarding the safety of DNA and vector-based vaccines, the use of antigen-encoding mRNA for vaccination approaches is increasingly gaining interest. mRNA-based vaccines present several advantages as compared with antigen delivery by viral vectors or plasmid DNA. First, mRNA cannot integrate in the host genome, therefore rendering its application much safer.1Pascolo S Vaccination with messenger RNA (mRNA).Handb Exp Pharmacol. 2008; 183: 221-235Crossref PubMed Scopus (108) Google Scholar Second, immunization with mRNA results in transient expression of the encoded protein, thus enabling a more controlled antigen exposure and minimizing the risk of tolerance induction. Third, additional sequences such as plasmid backbone or viral promotors are lacking. Finally, RNA does not need to cross the nuclear barrier for protein expression and therefore offers the possibility to transfect slow or nondividing cells.2Bettinger T Carlisle RC Read ML Ogris M Seymour LW Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells.Nucleic Acids Res. 2001; 29: 3882-3891Crossref PubMed Scopus (187) Google Scholar Although intranodal delivery of naked mRNA has been shown to elicit potent CD4+ and CD8+ T cell responses in mouse models,3Kreiter S Selmi A Diken M Koslowski M Britten CM Huber C et al.Intranodal vaccination with naked antigen-encoding RNA elicits potent prophylactic and therapeutic antitumoral immunity.Cancer Res. 2010; 70: 9031-9040Crossref PubMed Scopus (215) Google Scholar,4Kreiter S Diken M Selmi A Diekmann J Attig S Hüsemann Y et al.FLT3 ligand enhances the cancer therapeutic potency of naked RNA vaccines.Cancer Res. 2011; 71: 6132-6142Crossref PubMed Scopus (55) Google Scholar,5Van Lint S Goyvaerts C Maenhout S Goethals L Disy A Benteyn D et al.Preclinical evaluation of TriMix and antigen mRNA-based antitumor therapy.Cancer Res. 2012; 72: 1661-1671Crossref PubMed Scopus (142) Google Scholar extranodal administration of naked mRNA appears to be more challenging because of its susceptibility to extracellular nucleases. Although some studies have reported the induction of immune responses in mice in response to non-nodal parenteral administration of naked mRNA,6Wolff JA Malone RW Williams P Chong W Acsadi G Jani A et al.Direct gene transfer into mouse muscle in vivo.Science. 1990; 247: 1465-1468Crossref PubMed Scopus (3230) Google Scholar,7Conry RM LoBuglio AF Wright M Sumerel L Pike MJ Johanning F et al.Characterization of a messenger RNA polynucleotide vaccine vector.Cancer Res. 1995; 55: 1397-1400PubMed Google Scholar,8Pulido MR Sobrino F Borrego B Sáiz M RNA immunization can protect mice against foot-and-mouth disease virus.Antiviral Res. 2010; 85: 556-558Crossref PubMed Scopus (7) Google Scholar these findings have been contradicted by others.3Kreiter S Selmi A Diken M Koslowski M Britten CM Huber C et al.Intranodal vaccination with naked antigen-encoding RNA elicits potent prophylactic and therapeutic antitumoral immunity.Cancer Res. 2010; 70: 9031-9040Crossref PubMed Scopus (215) Google Scholar,9Martinon F Krishnan S Lenzen G Magné R Gomard E Guillet JG et al.Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA.Eur J Immunol. 1993; 23: 1719-1722Crossref PubMed Scopus (274) Google Scholar,10Hoerr I Obst R Rammensee HG Jung G In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies.Eur J Immunol. 2000; 30: 1-7Crossref PubMed Scopus (200) Google Scholar Nonviral carriers such as cationic polymers and cationic lipids have been used successfully to stabilize antigen-encoding DNA, thus increasing transfection efficiency and enhancing immune responses in vivo.11Jafari M Soltani M Naahidi S Karunaratne DN Chen P Nonviral approach for targeted nucleic acid delivery.Curr Med Chem. 2012; 19: 197-208Crossref PubMed Scopus (102) Google Scholar In addition, such particulate delivery systems target antigen-specific cells (APCs) therefore resulting in enhanced antigen presentation and subsequent induction of immune responses.12De Koker S Lambrecht BN Willart MA van Kooyk Y Grooten J Vervaet C et al.Designing polymeric particles for antigen delivery.Chem Soc Rev. 2011; 40: 320-339Crossref PubMed Scopus (116) Google Scholar Nonetheless, few studies have explored this approach to increase delivery and effectiveness of mRNA vaccines in vivo. As a result, knowledge concerning potency of nonviral delivery of mRNA vaccines is scarce and data describing its immunological properties are largely lacking.9Martinon F Krishnan S Lenzen G Magné R Gomard E Guillet JG et al.Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA.Eur J Immunol. 1993; 23: 1719-1722Crossref PubMed Scopus (274) Google Scholar,10Hoerr I Obst R Rammensee HG Jung G In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies.Eur J Immunol. 2000; 30: 1-7Crossref PubMed Scopus (200) Google Scholar,13Scheel B Aulwurm S Probst J Stitz L Hoerr I Rammensee HG et al.Therapeutic anti-tumor immunity triggered by injections of immunostimulating single-stranded RNA.Eur J Immunol. 2006; 36: 2807-2816Crossref PubMed Scopus (91) Google Scholar,14Lu D Benjamin R Kim M Conry RM Curiel DT Optimization of methods to achieve mRNA-mediated transfection of tumor cells in vitro and in vivo employing cationic liposome vectors.Cancer Gene Ther. 1994; 1: 245-252PubMed Google Scholar In this study, we have demonstrated the feasibility of using antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE) as a novel immunization strategy capable of evoking functional T cell responses. Furthermore, by studying the interaction between mRNA/carrier and dendritic cells, we have gained surprising insights regarding the role of type I interferons (IFNs) in modulating the mRNA-based immune response. These findings are of high relevance for the further design and development of RNA based vaccines, and will pave the way toward improved mRNA vaccination approaches. In this study, two types of nonviral carriers were evaluated: the polymers poly(β-amino ester) and in vivo jetPEI, and the cationic lipids Lipofectamine 2000 and DOTAP/DOPE. Cationic polymers consist of repeating units of chemical or natural monomers and condense with nucleic acids to form rigid, colloidal nanocomplexes. In contrast, cationic lipids assemble into dynamic spherical vesicles composed of phospholipid bilayers surrounding an aqueous compartment. To determine whether complexation of mRNA by any of these carriers could augment antigen-specific immune responses, mice were immunized with 20 µg of gag mRNA complexed with polymers or cationic lipids. Splenocytes and lymph node cells were isolated 3 weeks after immunization and restimulated with Gag peptides in an IFN-γ enzyme-linked immunosorbent spot (ELISPOT). Gag-specific IFN-γ secreting T cells were observed in spleens and lymph nodes of mice immunized with gag mRNA complexed with the cationic lipids Lipofectamine 2000 and DOTAP/DOPE, but not in mice immunized with naked mRNA or polymer-complexed mRNA (Figure 1a). In addition, DOTAP/DOPE complexed mRNA was capable of evoking IFN-γ secreting T cells to a similar extent as DOTAP/DOPE complexed DNA, suggesting mRNA-based vaccines might equal the potency of DNA based vaccines when stabilized properly (Supplementary Figure S1 online). For the subsequent detailed analysis of the T cell stimulatory capacity of antigen-encoding mRNA, DOTAP/DOPE was used to complex gag mRNA as this carrier induced superior T-cell responses. Because the activation of naive T cells constitutes the first step in adaptive immunity, we evaluated the expression of the early activation marker CD69 on T cells in the draining lymph nodes of mice injected in the footpad with 4 µg of DOTAP/DOPE-complexed gag mRNA (DOTAP gag). The popliteal lymph nodes were isolated 24 hours later, and the number of CD69+ cells within gated CD4+ and CD8+ T cells was determined by flow cytometry. Our results show that there was a significant increase in the number of CD69+ T cells in the lymph nodes of mice receiving DOTAP gag as compared with DOTAP/DOPE-injected control mice (DOTAP) or mice receiving naked gag (Figure 1b). Induction of strong cytotoxic T cell responses is considered one of the prerequisites for the development of successful therapeutic vaccines against cancer and HIV. To address whether DOTAP/DOPE complexed mRNA-based immunization was capable of inducing functional “killer” responses, we performed an in vivo cytotoxicity assay. Immunization with DOTAP gag resulted in a significant increase of Gag-specific killing as compared with immunization with naked gag or DOTAP/DOPE alone (Figure 1c). The potency of DOTAP gag-induced CD4 T-helper response to stimulate B cell immunity was evaluated by analyzing the induction of germinal center (GC) B cells. During the development of T cell-dependent antibody responses, GCs are formed by proliferating B cells and provide the main source of memory B cells and long-living plasma cells.15Klein U Dalla-Favera R Germinal centres: role in B-cell physiology and malignancy.Nat Rev Immunol. 2008; 8: 22-33Crossref PubMed Scopus (626) Google Scholar The number of GC cells (CD3− CD19+ GL-7+ CD95+) within the lymph node B cell population (CD3− CD19+) was quantified 7 days after the administration of DOTAP gag by flow cytometry. We observed a significant increase in GC cells as compared to mice receiving naked gag or DOTAP control (Figure 1d,e). In conclusion, these findings demonstrate that immunization with DOTAP/DOPE-complexed antigen-encoding mRNA induces antigen-specific, functional T cells resulting in potent cytotoxic T lymphocytes and GC formation. The use of prime-boost strategies, in which prime and boost immunization consist of different vaccine modalities, has been shown to efficiently increase the potency of both DNA and vector-based vaccines. To evaluate whether this principle also applies to mRNA immunization, a protein boost with the HIV-1 capsid protein p24 (processed from the Gag polyprotein) was administered subcutaneously to DOTAP gag primed mice. Boosting with the p24 protein resulted in an over sevenfold increase of antigen-specific IFN-γ secreting cells in both spleens and lymph nodes of DOTAP gag immunized mice (Figure 2a). In addition, induction of antigen-specific interleukin (IL)-2 production was observed in mice receiving DOTAP gag + p24, but not in mice receiving DOTAP gag alone. Surprisingly, Gag-specific IL-2 secreting T cells were also detected in lymph nodes of mice receiving only p24 protein, without DOTAP gag priming. Next, we determined whether the p24 protein boost could induce p24-specific humoral responses. A serum enzyme-linked immunosorbent assay was performed to assess the levels of p24-specific immunoglobulin (Ig)M, IgG1, and IgG2c antibodies. Of note, immunization with DOTAP gag plus p24 led to relatively high antibody titers, whereas naked gag plus p24 (which was able to induce IL-2) failed to induce p24-specific antibodies (Figure 2b). Serum levels of IgG1 were relatively low in comparison with IgG2c, thereby indicating a preference for Th1-dependent class switching. Vaccine-elicited effector memory T cells that migrate from the lymphoid site of immune induction to the peripheral site of pathogen entry where they can exert their effector functions without requiring preceding rounds of antigen stimulation and cell division in the lymph nodes, might be of great importance to eliminate infection very early upon pathogen entry. Therefore, the induction of such effector memory T cells might be crucial for the development of an effective vaccine against insidious pathogens such as HIV.16Jameson SC Masopust D Diversity in T cell memory: an embarrassment of riches.Immunity. 2009; 31: 859-871Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar To address the capacity of parenteral mRNA-based vaccination in evoking the recruitment of effector T cells toward peripheral (mucosal) tissues, CD4 and CD8 T cells were isolated from the lungs one month following the subcutaneous booster immunization and subsequently restimulated with Gag peptides in an IFN-γ ELISPOT assay. Remarkably, even without preceding pulmonary antigen challenge, high levels of Gag-responsive T cells were present in the lung, capable of rapidly secreting IFN-γ upon antigenic stimulation. Although low levels CD4 T cell responses were detectable, T cell responses appeared to be largely limited to the CD8 T cell compartment, a phenomenon easily anticipated given the need for endosomal mRNA escape and subsequent cytosolic expression of the encoded antigens, which are consequently presented via major histocompatibility complex (MHC)-I. Administration of a local pulmonary p24 protein boost dramatically increased the level of the CD4 T cell response (40-fold), and even further amplified the CD8 T cell response, albeit to a less degree. Whether these amplified T cell responses are a result from local antigen presentation and T cell proliferation, or whether they represent newly recruited T cells from the draining lymph nodes remains to be determined. In any case, delivering a protein boost appears to dramatically amplify the CD4 T cell response, a feature that can be explained by the mainly MHC-II mediated route of antigen presentation of soluble antigens. Given the role of CD4 T cells in supporting CD8 T cell function, the administration of a protein boost might significantly enhance the quality and longevity of the mRNA evoked CD8 T cell response. Most traditional vaccines consisting of recombinant protein antigens fail to activate pattern recognition receptors, therefore requiring the addition of adjuvants to evoke effector responses.17Guy B The perfect mix: recent progress in adjuvant research.Nat Rev Microbiol. 2007; 5: 505-517Crossref PubMed Scopus (489) Google Scholar In this respect, the use of mRNA vaccines may confer an extra advantage as in addition to encoding the antigen, DOTAP/DOPE complexed mRNA might also display intrinsic adjuvant capacity, as dendritic cells (DCs) express various pattern recognition receptors including TLR3 and TLR7/8 that recognize respectively double stranded RNA and single stranded RNA.18Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4907) Google Scholar,19Diebold SS Kaisho T Hemmi H Akira S Reis e Sousa C Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.Science. 2004; 303: 1529-1531Crossref PubMed Scopus (2725) Google Scholar A first indication that DOTAP/DOPE complexed mRNA entails intrinsic adjuvant properties came from assessing the maturation status of bone marrow-derived DCs transfected with 2.5 µg of DOTAP gag. As depicted in Figure 3a, successfully transfected cells (Gag+ DCs) expressed elevated levels of the co-stimulatory markers CD40, CD80, and CD86 as compared with their nontransfected counterparts (Gag- DCs). Moreover, quantitative real-time reverse transcript PCR (RT-qPCR) analysis of DCs incubated with DOTAP gag showed a modest increase in expression levels of the proinflammatory cytokines IL-6 and IL-1β, and a profound upregulation of the type I IFNs IFN-α and IFN-β (Figure 3b). This massive increase in expression levels of type I IFN was not observed in DCs exposed to naked gag or DOTAP alone. These findings were confirmed by studying the nuclear translocation of the IFN regulatory factors (IRF) IRF-3 and IRF-7 using confocal microcopy. Upon activation, IRF-3/7 dimerizes and translocates to the nucleus to activate transcription of type I IFN genes. As shown in Figure 3c,d, both IRF-3 and IRF-7 rapidly migrated to the nucleus after in vitro stimulation of DC with DOTAP gag. To evaluate to what extent the strong type I IFN response to DOTAP/DOPE complexed mRNA was dependent on TLR3 and/or TLR7 mediated signaling, we compared expression levels of type IFN in bone marrow-derived DCs isolated from MyD88−/− (defective in TLR7 signaling), TIR-domain-containing adapter-inducing interferon-β (TRIF)−/− (defective in TLR3 signaling) and wild-type (WT) mice. Although MyD88 deficiency had very little impact on type I IFN expression, TRIF deficiency in contrast evoked a relatively strong decrease in IFN-α expression levels and a moderate downregulation of IFN-β expression (Figure 3e). The capacity of DOTAP/DOPE complexed mRNA to induce type I IFN was also confirmed in vivo by performing enzyme-linked immunosorbent assay assays on serum isolated 1, 3, 6, and 24 hours after i.v. administration of 20 µg DOTAP gag. We observed a transient induction of IFN-α, with serum levels peaking at 3–6 hours (Figure 3f). Furthermore, footpad injection of DOTAP gag resulted in a rapid, but transient influx of inflammatory monocytes (MHC-II+ Ly6Chi CD11bhi cells) to the draining lymph nodes (Figure 3g). Since the recruitment of inflammatory monocytes is typically observed following microbial infection or adjuvant injection, these data confirm the intrinsic immune-stimulatory properties of DOTAP/DOPE complexed mRNA.20Nakano H Lin KL Yanagita M Charbonneau C Cook DN Kakiuchi T et al.Blood-derived inflammatory dendritic cells in lymph nodes stimulate acute T helper type 1 immune responses.Nat Immunol. 2009; 10: 394-402Crossref PubMed Scopus (273) Google Scholar Although type I IFNs are considered to be important promoters of cellular immunity,21Brinkmann V Geiger T Alkan S Heusser CH Interferon alpha increases the frequency of interferon gamma-producing human CD4+ T cells.J Exp Med. 1993; 178: 1655-1663Crossref PubMed Scopus (438) Google Scholar,22Havenar-Daughton C Kolumam GA Murali-Krishna K Cutting Edge: The direct action of type I IFN on CD4 T cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection.J Immunol. 2006; 176: 3315-3319Crossref PubMed Scopus (166) Google Scholar,23Kolumam GA Thomas S Thompson LJ Sprent J Murali-Krishna K Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection.J Exp Med. 2005; 202: 637-650Crossref PubMed Scopus (717) Google Scholar,24Le Bon A Durand V Kamphuis E Thompson C Bulfone-Paus S Rossmann C et al.Direct stimulation of T cells by type I IFN enhances the CD8+ T cell response during cross-priming.J Immunol. 2006; 176: 4682-4689Crossref PubMed Scopus (217) Google Scholar their induction might interfere with the potency of mRNA-based vaccines by inducing antiviral responses resulting in break-down of exogenous mRNA and inhibition of translation.25Pichlmair A Reis e Sousa C Innate recognition of viruses.Immunity. 2007; 27: 370-383Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar To address these issues, IFNαR−/− mice were immunized with DOTAP gag. Surprisingly, we observed a strong increase in the levels IFN-γ and IL-2 secreting T cells as compared with WT mice, suggesting a rather negative effect of type I IFN (Figure 4a). To investigate whether this effect might indeed be due to type I IFN-mediated inhibition of antigen expression, we compared efficiency of Gag expression following DOTAP gag transfection in WT and IFNαR−/− DCs. As depicted in Figure 4b, the frequency of Gag positive DCs was clearly elevated in IFNαR−/− DCs. Similar results were obtained with egfp mRNA (Supplementary Figure S2 online), suggesting that this phenomenon is independent of the specific mRNA sequence of Gag. MyD88−/− DCs showed no increase in Gag expression, which is in agreement with the abovementioned finding that expression of type I IFN is not affected in MyD88−/− DCs. Although Gag expression did not reach the same level as in IFNαR−/− DCs, TRIF−/− DCs showed a clear increase in Gag expression as compared to WT cells. Again, these data are in accordance with the earlier observed intermediate decrease in type I IFN in TRIF−/− DCs, indicating that DOTAP gag-induced expression of type I IFN is partially dependent on TLR3 signaling. Although the capacity of type I IFN to link innate to adaptive immunity has been shown to be crucial for the adjuvant activity of TLR3 and TLR7 agonists,26Longhi MP Trumpfheller C Idoyaga J Caskey M Matos I Kluger C et al.Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant.J Exp Med. 2009; 206: 1589-1602Crossref PubMed Scopus (487) Google Scholar,27Rajagopal D Paturel C Morel Y Uematsu S Akira S Diebold SS Plasmacytoid dendritic cell-derived type I interferon is crucial for the adjuvant activity of Toll-like receptor 7 agonists.Blood. 2010; 115: 1949-1957Crossref PubMed Scopus (95) Google Scholar they clearly interfere with the potency of mRNA-based vaccination. Nevertheless, activation of innate immune responses is a critical prerequisite for all vaccines to induce adaptive immune responses. To determine to what extent IFNαR signaling is required for the DOTAP gag-mediated activation of innate immunity, we evaluated the capacity of DOTAP gag to activate IFNαR−/− DCs and to promote the recruitment of inflammatory monocytes following subcutaneous injection in IFNαR−/− mice. Similar to WT cells, we observed an upregulation of the maturation markers CD40 and CD86 (but not CD80) on DOTAP gag-transfected IFNαR−/− bone marrow-derived DCs (Figure 4c). In addition, subcutaneous injection of DOTAP/DOPE complexed mRNA still resulted in the infiltration of inflammatory monocytes in the draining lymph nodes (Figure 4d). When compared with WT mice injected with DOTAP gag, total numbers of recruited inflammatory monocytes were lower in IFNαR−/− mice, a phenomenon which might however be at least partially explained by the basal lower counts of DCs and monocytes in the lymph nodes of IFNαR−/− mice. Taken together, these results demonstrate that IFNαR signaling is not required for the innate immune activation and recruitment of DCs in DOTAP gag immunized mice. In recent years, antigen-encoding mRNA has emerged as a promising alternative to classical DNA and viral vector-based immunization approaches. mRNA vaccines have sparked interest particularly in the field of tumor vaccination, as these strategies hold the potential to introduce a broad spectrum of patient-specific tumor associated antigens.28Bringmann A Held SA Heine A Brossart P RNA vaccines in cancer treatment.J Biomed Biotechnol. 2010; 2010: 623687Crossref PubMed Scopus (39) Google Scholar,29Kreiter S Diken M Selmi A Türeci ö Sahin U Tumor vaccination using messenger RNA: prospects of a future therapy.Curr Opin Immunol. 2011; 23: 399-406Crossref PubMed Scopus (107) Google Scholar Moreover, the use of mRNA vaccines could also be beneficial in the context of therapeutic HIV vaccination as it enables the delivery of multiple HIV genes and their mutations, thus allowing the approximate patient-specific representation of the quasi species of the virus.30Van Gulck E Van Tendeloo VF Berneman ZN Vanham G Role of dendritic cells in HIV-immunotherapy.Curr HIV Res. 2010; 8: 310-322Crossref PubMed Scopus (16) Google Scholar In this work, we have evaluated the potency of an mRNA vaccine complexed with the cationic lipid DOTAP/DOPE in an in vivo preclinical mouse model. In addition, we have investigated the mechanisms contributing to the immunogenicity of DOTAP/DOPE mediated delivery of mRNA. Finally, we have studied the role of type I IFN in the induction of immune responses against the encoded antigen. In a first set of experiments, we assessed the capacity of different nonviral carriers to induce antigen-specific immune responses against mRNA encoding the HIV-1 protein Gag. Our results identified DOTAP/DOPE as a potent carrier for subcutaneous delivery of mRNA to induce immune responses, a feature which could not be achieved using naked mRNA. DOTAP/DOPE mRNA vaccination appeared to strongly evoke CD8 T cells, which were fully capable of recognizing and killing peptide-pulsed target cells in vivo. Of note, subcutaneous immunization allowed the emigration of effector T cells to nonlymhoid tissues, as was demonstrated by the high numbers of Gag-responsive CD8 T cells in the lungs of DOTAP/DOPE-complexed gag mRNA immunized mice. The presence of high numbers of such effector memory cells at the site of pathogen entry might be of significant importance for the development of effective vaccines against insidious pathogens that rapidly spread or evade immunity. Further studies are needed to address whether such CD8 T cells are also present in the gastrointestinal and/or genital tract, the sites of HIV entry. As could be anticipated by the cytosolic expression of the mRNA encoded antigen, mRNA-based vaccination mainly promotes the induction of CD8 T cell responses. Nevertheless, low levels of antigen-specific CD4 T cells were present in the pulmonary compartment, and their numbers could be dramatically increased by delivering a protein boost, which also strongly amplified the T cell response in the lymphoid compartment and enabled the generation of a Th1-skewed humoral immune response. During the last decade, it has become increasingly clear that the induction of effector T- and B cell responses largely depends on the initial activation of the innate immune system via a set of pattern recognition receptors. Given the robust induction of T- and B cell responses observed in response to lipid-based mRNA vaccination, we speculated that DOTAP/DOPE complexed mRNA may act as a viral mimic by triggering RNA recognizing pattern recognition receptors in the endosomes or cytosol of DCs. We showed that bone marrow-derived DCs upregulated CD40 and CD86 in response to DOTAP gag, indicating activation of DCs. Moreover, we observed a moderate increase of IL-6, IL-1β, and a profound induction of type I IFN, a cytokine typically released in response to viral RNA.31Baum A García-Sastre A Induction of type I interferon by RNA viruses: cellular receptors and their substrates.Amino Acids. 2010; 38: 1283-1299Crossref PubMed Scopus (102) Google Scholar Data obtained from TRIF knockout DCs indicated that DOTAP gag-induced DC activation is partially dependent on TLR3 signaling. However, it is likely that other signaling pathways are also involved, as endosomal escape of lipoplexes may enable activation of cytosolic RNA sensors. The intrinsic capacity of DOTAP/DOPE complexed antigen-encoding mRNA to activate the innate immune system was also confirmed in vivo by the systemic release of type I IFN and the rapid recruitment of inflammatory monocytes to the draining lymph nodes of DOTAP gag immunized mice. The induction of type I IFN in response to antigen-encoding mRNA is possibly a double edged sword. Although systemic production of type I IFN has been s" @default.
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• W2026083914 title "Type I IFN Counteracts the Induction of Antigen-Specific Immune Responses by Lipid-Based Delivery of mRNA Vaccines" @default.
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• W2026083914 cites W1709368598 @default.
• W2026083914 cites W1979289100 @default.
• W2026083914 cites W1994391392 @default.
• W2026083914 cites W2014495927 @default.
• W2026083914 cites W2025980739 @default.
• W2026083914 cites W2026336857 @default.
• W2026083914 cites W2026485598 @default.
• W2026083914 cites W2035983102 @default.
• W2026083914 cites W2036710997 @default.
• W2026083914 cites W2040088522 @default.
• W2026083914 cites W2045121415 @default.
• W2026083914 cites W2061867251 @default.
• W2026083914 cites W2061899137 @default.
• W2026083914 cites W2069390230 @default.
• W2026083914 cites W2072874308 @default.
• W2026083914 cites W2076361525 @default.
• W2026083914 cites W2083407483 @default.
• W2026083914 cites W2083958143 @default.
• W2026083914 cites W2096109794 @default.
• W2026083914 cites W2101578637 @default.
• W2026083914 cites W2113326511 @default.
• W2026083914 cites W2125677437 @default.
• W2026083914 cites W2126082800 @default.
• W2026083914 cites W2131179322 @default.
• W2026083914 cites W2150098469 @default.
• W2026083914 cites W2155991561 @default.
• W2026083914 cites W2156313733 @default.
• W2026083914 cites W2168008865 @default.
• W2026083914 cites W4241462697 @default.
• W2026083914 cites W81585652 @default.
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